title: Inference of differentiation time for single cell transcriptomes using cell population reference data

city: Shanghai

first: Jing-Dong

middle: Jackie

last: Han

address: 320 Yueyang Road

institute: CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

summary: To understand the molecular mechanisms of neural commitment from mouse ESCs, we selected very dense time points during neural differentiation based on qPCR results and performed mRNA-sequencing at both of cell population and single cell levels.

overall_design: Two neural induction protocols were used. 21 time points were selected for cell population mRNA-seq. Of them, 8 time points were selected for single cell RNA-seq, and for each time point, 8 individual cells were selected.

first: Jing

middle: D


title: A novel Myb transcription factor represses conidiation and cephalosporin production in Acremonium chrysogenum

status: Public on Nov 28 2017

city: beijing

first: wang

last: ying

address: beichen

country: China

institute: State Key Laboratory of Mycology

type: Expression profiling by high throughput sequencing

summary: Acremonium chrysogenum is the industrial producer of cephalosporin C. We isolated a mutant (AC554) from a T-DNA inserted library of A. chrysogenum. AC554 exhibited reduced conidiation and lack of cephalosporin C production. In consistent, the transcription of cephalosporin biosynthetic genes pcbC and cefEF was obviously decreased in AC554. TAIL-PCR and sequence analysis indicated that a T-DNA was inserted in the upstream of an open reading frame (ORF) which was designated AcmybA. Sequence analysis indicated that AcmybA encodes a novel Myb domain containing transcriptional factor. Observation of red fluorescence protein (RFP) tagged AcMybA showed that AcMybA is naturally located in the nuclear of A. chrysogenum. Transcription analysis demonstrated that AcmybA was overexpressed in AC554. In contrast with AC554, the AcmybA deleted mutant (DAcmybA) overproduced conidia in LPE medium and increased cephalosporin production during fermentation. To determine the genes under the influence of AcmybA, we sequenced and compared the transcriptome of DAcmybA, AC554 and the wild-type strain at different developmental stages. Results confirmed the repression of AcMybA on the key conidiation regulatory gene AcbrlA and the cephalosporin biosynthetic genes. Among the targets of AcMybA, 10 putative regulatory genes were selected and overexpressed in A. chrysogenum. Taken together, our results indicate that AcMybA negatively regulates conidiation and cephalosporin production in A. chrysogenum.

overall_design: Nine samples were analyzed by RNA-seq including the WT, ∆AcmybA and AcmybAOE with three time points 48, 72 and 96 hour in the LPE medium.

first: Ying

last: Wang

first: Pengjie

last: Hu

first: Liang-kun

last: Long

first: Kuan

last: Li

first: Xiaoling

last: Zhang

first: Yuanyuan

last: Pan

first: Gang

last: Liu


title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

city: Kaohsiung

first: Yi-Jen

last: Chen

address: 100, Shih-Chuan 1st Road

department: Graduate Institute of Clinical Medicine, College of Medicine

country: Taiwan

institute: Kaohsiung Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: The role of osteoblasts in peri-articular bone loss and bone erosion in rheumatoid arthritis (RA) has gained much attention, and microRNAs are hypothesized to play critical roles in the regulation of osteoblast function in RA. The aim of this study is to explore novel microRNAs differentially expressed in RA osteoblasts and to identify genes potentially involved in the dysregulated bone homeostasis in RA. RNAs were extracted from cultured normal and RA osteoblasts for sequencing. Using the next generation sequencing and bioinformatics approaches, we identified 35 differentially expressed microRNAs and 13 differentially expressed genes with potential microRNA–mRNA interactions in RA osteoblasts. The 13 candidate genes were involved mainly in cell–matrix adhesion, as classified by the Gene Ontology. Two genes of interest identified from RA osteoblasts, A-kinase anchoring protein 12 (AKAP12) and leucin rich repeat containing 15 (LRRC15), were found to express more consistently in the related RA synovial tissue arrays in the Gene Expression Omnibus database, with the predicted interactions with miR-183-5p and miR-146a-5p, respectively. The Ingenuity Pathway Analysis identified AKAP12 as one of the genes involved in protein kinase A signaling and the function of chemotaxis, interconnecting with molecules related to neovascularization. The findings indicate new candidate genes as the potential indicators in evaluating therapies targeting chemotaxis and neovascularization to control joint destruction in RA.

overall_design: Next-generation sequencing for mRNA and small RNA of normal and RA osteoblasts

first: Wei-An

last: Chang

first: Ya-Ling

last: Hsu

first: Chia-Hsin

first: Po-Lin

last: Kuo


title: Boundary Formation is an Inherent Property of Mobile Small RNA Gradients

status: Public on Dec 01 2017

city: Cold Spring Harbor

first: Damianos

last: Skopelitis

state: NY

address: One Bungtown Rd

department: Cancer Biology

country: USA

institute: Cold Spring Harbor Laboratory

summary: Small RNAs recently emerged as a new class of mobile instructive signals in development. Here, we investigate their mechanism of action and show that the gradients formed by mobile small RNAs generate sharply defined domains of target gene expression. By modulating the source of artificial miRNAs we show that boundary formation is an inherent property of the small RNA gradient itself. The threshold-based readout of such gradients is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. In addition to generating sharp expression domains of their immediate targets, the readouts of opposing small RNA gradients enable formation of stable and uniformly positioned developmental boundaries. These novel patterning properties of small RNAs are reminiscent of those of morphogens in animal systems. However, their exceptionally high specificity, direct mode of action, and the fully intrinsic nature of their gradients, distinguish mobile small RNAs from classical morphogens. Our findings present mobile small RNAs and their targets as highly portable and evolutionarily-tractable regulatory modules through which to create pattern in development and beyond.

overall_design: Examination of artificial miRNA (miRGFP) expression and GFP-derived secondary siRNA production, in p35S:miRGFP and control p35S:3xNLS-GFP (no miRGFP) Arabidopsis seedlings, in an rdr6 null mutant background impaired for siRNA production.

first: Anna

last: Benkovics

first: Aman

last: Husbands

first: Marja

last: Timmermans


title: The transcriptome effect of knocking down EZH2 in TamR MCF7L

city: San Antonio

first: Kexin

last: Xu

state: Texas

address: 7703 Floyd Curl, MC 8257

department: Molecular Medicine

institute: UT Health Science Center at San Antonio

summary:
Purpose: Increasing evidence suggests that epigenetic reprogramming contributes significantly to the development of endocrine therapy resistance in breast cancer. The goal of this work is to explore how the histone methyltransferase EZH2 interacts with ER signaling and drives the insensitiveness of breast cancer cells to the antagonistic effect of tamoxifen on ER activity. Therefore,  we comprehensively analyzed the transcriptional program regulated by EZH2 in tamoxifen-resistant (TamR) MCF-7 cells.
Methods: TamR MCF-7 cells between passage 142-144 upon  were used for this assay. For mRNA-Seq, cells are transfected with scrambled control shRNAs (shCtrl) or shRNAs targeting EZH2 (shEZH2). Total RNA were extracted by TRIzol (Invitrogen) and libraries were constructed using Illumina TruSeq RNA Sample Prep Kit v2 (Cat.# RS-122-2001). Hiseq 3000 was used for sequencing.

overall_design: Transcriptome profiles of Tamoxifen resistance MCF-7 infected with shCtrl or shEZH2 were sequenced in duplicate using Illumina HiSeq 3000.

first: Yanming

last: Wu

first: Zhao


title: Cockayne syndrome A and B proteins regulate the transcription arrest upon genotoxic stress through a ubiquitin/proteasome degradation process

city: Illkirch

first: Tao

last: YE

address: 1 rue Laurent Fries

country: France

institute: IGBMC (CNRS/INSERM/UDS)

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Hippocampus and hypothalamus RNA-sequencing of WFS1-deficient mice [hippocampus]

city: Perth

first: Sulev

last: Koks

state: Western Australia

address: 90 South Street

department: Centre for Molecular Medicine and Innovative Therapeutics

country: Australia

institute: Murdoch University

summary: Wolfram syndrome is caused by mutations in the WFS1 gene. WFS1 protein dysfunction results in a range of neuroendocrine syndromes and is mostly characterized by juvenile-onset diabetes mellitus and optic atrophy. WFS1 has been shown to participate in membrane trafficking, protein processing and Ca2+ homeostasis in the endoplasmic reticulum.  In the present study we aimed to find the transcriptomic changes influenced by Wfs1 in the hypothalamus and hippocampus using RNA-sequencing. We used WFS1-deficient mice as a model system to analyze the changes in transcriptional networks.  The number of differentially expressed genes between hypothalami of WFS1-deficient (Wfs1KO) and wild-type (WT) mice was 43 and between hippocampi 311 with False Discovery Rate (FDR) <0.05. In hypothalamus of Wfs1KO mice one of the most upregulated genes was Avpr1a whilst Avpr1b was significantly upregulated in hippocampus. Trpm8 was the most upregulated gene in the hippocampus of Wfs1KO mice. The functional analysis revealed significant enrichment of networks and pathways associated with protein synthesis, cell-to-cell signaling and interaction, molecular transport, metabolic disease and nervous system development and function.   In conclusion, the transcriptomic profiles of WFS1-deficient hypothalamus and hippocampus do indicate the activation of degenerative molecular pathways causing the clinical occurrences typical to Wolfram syndrome.

overall_design: 8 samples: twp genotypes, 4 individuals in each genotype

first: Marilin

last: Ivask

first: Sander

last: Pajusalu

first: Ene

last: Reimann

last: Kõks


title: Transcriptional response to type I IFN in bat P. alecto cells

city: Dallas

first: Pamela

middle: C

last: Ce La Cruz

state: TX

address: 6000 Harry Hines Blvd

department: Dept. Microbiology

institute: UT Southwestern Medical Center

summary: Bats are reservoirs of a number of highly pathogenic human viruses, yet they remain relatively asymptomatic during infection. Whether this viral resistance is due to a unique immune system is unknown. An evolutionarily conserved feature of vertebrate antiviral immunity is the interferon (IFN) response, which triggers cellular defenses through interferon-stimulated gene (ISG) expression. While bats encode an intact IFN system, global ISG expression patterns in bat cells are not well characterized.  Here, we used RNA-Seq to assess the transcriptional response to IFNα in cells derived from the bat Pteropus alecto (black flying fox). We show induction of more than 100 transcripts, most of which are canonical ISGs observed in other species. Kinetic gene profiling revealed that P. alecto ISGs fall into two unique temporal subclusters with similar early induction kinetics but distinct late-phase declines.  In contrast to bat ISGs, human ISGs generally remained elevated for longer periods following IFN treatment, suggesting host-based differences in gene regulatory mechanisms. Notably, we also identified a small group of non-canonical bat ISGs, including an enzymatically active RNASEL. These studies provide insight into the innate immune response of an important viral reservoir and lay a foundation for studies into the immunological features that may underlie a unique virus-host relationship in bats.

overall_design: Time course in type I IFN-treated cells derived from the bat P. alecto using 5 time points and biological triplicates.

last: De La Cruz-Rivera

first: Mohammed

last: Kanchwala

first: Hanquan

last: Liang

first: Ashwani

last: Kumar

first: Linfa

last: Wang

first: Chao

last: Xing

first: John

middle: W

last: Schoggins


title: Allergen-specific immunotherapy modulates the balance of circulating Tfh and Tfr cells

city: La Jolla

first: Bjoern

last: Peters

address: 9420 Athena Cir

institute: La Jolla Institute for Allergy and Immunology

summary: Allergen-specific immunotherapy has major effects on the frequency of peripheral Tfh and Tfr cells, which may be regulated by IL-2

overall_design: RNA-sequencing of sorted memory CXCR5 high and CXCR5low T cells from cryopreserved PBMC of 5 subjects with allergy  and 5 patients undergoing allergen-specific immunotherapy

first: Veronique

last: Schulten

first: Victoria

last: Tripple

first: Gregory

last: Seumois

first: Yian

last: Qian

first: Richard

last: Scheuermann

first: Zheng

last: Fu

first: Michela

last: Locci

first: Sandy

last: Rosales

first: Pandurangan

last: Vijayanand

first: Alessandro

last: Sette

first: Rafeul

last: Alam

first: Shane

last: Crotty


title: Inhibition of TGFβRI as a therapy for GATA4 deficient lung cancers [RNA-seq]

city: Beijing

first: huanwei

last: huang

address: 7 Science Park Rd., ZGC life science park

institute: NIBS

summary: GATA4 is frequently epigenetically silenced in lung cancers. However, the impact of GATA4 inactivation on tumorigenesis and related therapeutic strategy remain to be determined. Through the genome-wide screening of tumor suppressing transcription factors, we demonstrate that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vitro. Interestingly, ectopic GATA4 expression resulted in cellular senescence. Mechanistically, GATA4 up-regulates multiple miRNAs (miRNA-32, miRNA-301b, miR-320a, and miR-590) targeting TGFB2 mRNA and causes ensuing downregulation of WNT7B level to induce senescence. TGFBRI inhibitor synergizes with MEK1/2 inhibitor to promote lung cancer regression in Kras G12D/GATA4-/- mouse models. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGFB2 level and is significantly associated with poor prognosis.

overall_design: Compare the mRNA profiles in dox and vehicle  induced A549i cell by Illumina sequencing

first: Liang

last: Chen

first: Lei

last: Gao

first: Huanwei

last: Huang


title: Xiphophorus maculatus JP163 A RNA-Seq data for PacBio Genome Annotation (Nov.2017)

city: San Marcos

first: Yuan

last: Lu

address: university drive

institute: Xiphophorus Genetic Stock Center

summary: RNA from Xiphophorus maculatus (X. maculatus) are isolated and sequenced for annotation of enhanced X. maculatus genome

overall_design: 26 Xiphophorus maculatus (X. maculatus) fish at various developmental stages were sacrified for tissue specific RNA or whole fish RNA isolation, RNA-sequencing and for genome annotation.

first: Ronald

last: Walter

first: Manfred

last: Schartl

first: Wesly

last: Warren

first: William

last: Boswell

first: Mikki


title: An essential cell cycle regulator drives chromatin condensation in maturing erythroblasts

city: Rochester

first: Laurie

middle: A

last: Steiner

address: 601 Elmwood Avenue

department: Center for Pediatric Biomedical Research

institute: University of Rochester

summary: We report that Setd8 is essential for murine erythropoeisis. Setd8 null erythroblasts have severe defects in cell cycle progression, chromatin condensation,and heterochromatin integrity. They also have dysregulated gene expression.

overall_design: Ter119+,CD71+ Setd8 null and control erythroblasts were sorted from the fetal blood of E10.5 embryos. RNA from those erythroblasts was sent for RNA-seq.

first: Jeffrey

last: Malik

first: Jacquelyn

last: Lillis


title: Identification of RNA interacting with the sRNA sponge 3'ETSleuZ

city: SHERBROOKE

first: Eric

last: Massé

state: Québec

address: 3201, Jean Mignault

department: Biochemistry

country: Canada

institute: University of Sherbrooke

summary: During glyW-cysT-leuZ polycistronic tRNA maturation, the 3’external transcribed spacer (3’ETS) sequence is excised and act as a sRNA sponge (Lalaouna et al., 2015). Using MS2-affinity purification coupled with RNA sequencing (MAPS), we demonstrated that 3’ETSleuZ was highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs were shown to base pair with the same region in 3’ETSleuZ. Here, we use MS2-3’ETSleuZ as bait to co-purify all interacting sRNAs and confirm 3’ETSleuZ/RyhB and 3’ETSleuZ/RybB interactions.

overall_design: Identification of sRNAs co-purified with MS2-3'ETSleuZ. 3'ETSleuZ (without MS2) was used as control

first: David

last: Lalaouna


title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

status: Public on Nov 27 2017

city: Montréal

first: Claire

middle: Elizabeth

last: Nash

state: QC

address: 1001 Decarie Blvd

department: Urology

institute: Research Institute of McGill University Health Centre

summary: Prostate organogenesis involves epithelial growth in response to inductive signalling from specialised subsets of mesenchyme. To identify regulators and morphogens active in mesenchyme, we performed transcriptomic analysis using Tag-seq, RNA-seq, and single cell RNA-seq and defined new mesenchymal subsets and markers. We documented transcript expression using Tag-seq and RNA-seq in female rat Ventral Mesenchymal Pad (VMP) as well as adjacent urethra comprised of smooth muscle and peri-urethral mesenchyme. Transcripts enriched in VMP were identified in Tag-seq data from microdissected tissue, and RNA-seq data derived from cell populations and single cells. We identified 400 transcripts as enriched or specific to the VMP using bio-informatic comparisons of Tag-seq and RNA-seq data. Comparison with single cell RNA-seq identified transcripts yielded 45 transcriptscommon to both approaches. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue was comprised of two subgroups. Markers for these subgroups were highly subset specific. Thirteen transcripts were validated by qPCR to confirm cell specific expression in microdissected tissues, as well as expression in neonatal prostate. Immunohistochemical staining demonstrated that Ebf3 and Meis2 showed a restricted expression pattern in VMP condensed mesenchyme. Taken together, we demonstrate that the VMP shows limited cellular heterogeneity and that our high-resolution transcriptomic analysis identified new mesenchymal subset markers associated with prostate organogenesis.

overall_design: Tag-sequencing, RNA-sequencing and single-cell RNA-sequencing on 2 female inductive mesenchymal tissues of the developing prostate/urogenital tract.

middle: E

first: Axel

last: Thomson


title: Expression data from the chronic myeloid leukemia cell line K562 with or without AF1q/MLLT11 overexpressing

city: Jinan

first: Wei

last: Li

state: Shandong

address: #44,West Wenhua Road

institute: Shandong University

summary:
AF1q/MLLT11 expression was significantly upregulated in CML patients and elevated AF1q expression was associated with disease progression. Enforced AF1q expression promoted cell survival, protected CML cells from IM treatment and increased engraftment of CML cells in vivo.
We used microarrays in AF1q-overexpressing K562 cells and negative control cells to profile differentially expressed genes that might be involved in AF1q regulation.

overall_design: K562 cells infected with lentiviruses harboring AF1q were prepared in triplicates: AF1q-1,2,3, meanwhile negative control cells as RFP-1,2,3. 106 cells from each group were used for RNA extraction and hybridization on Affymetrix microarrays.

first: Min

last: Ji

first: Chunyan


title: ChIP-seq analysis of Rad21 binding during genome activation

city: Dunedin

first: Michael

last: Meier

address: 1st Floor Hercus Building, Dunedin School of Medicine, Great King Street

department: Pathology

country: New Zealand

institute: University of Otago

summary: The formation of a transcriptional competent zygote is a not fully understood process potentially involving changes in chromatin structure. Cohesin and CTCF are important nuclear architectural proteins that contribute to chromatin structure and gene regulation. Here we analyze the genome-wide location of Rad21 binding during zebrafish embryogenesis and show increased Rad21 binding over developmental time (2.5 hpf, 4.5 hpf , 10 hpf).

overall_design: ChIP-seq to generate genome wide binding data for Rad21 in three stages of zebrafish development, two biological replicates for each stage with input controls.

first: Julia

last: Horsfield


title: Development and characterization of a mouse epithelial cell monolayer platform (APECCS) of the small and large intestines

status: Public on Nov 30 2017

city: Fremont

first: Matthew

last: Siegel

state: CA

address: 34175 Ardenwood Blvd

institute: Ardelyx

summary: We describe the development and characterization of a mouse epithelial cell monolayer platform of the small and large intestines with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages.

overall_design: We established monolayer cultures were with the growth factor combination used for three-dimensional mouse colon organoids i.e. Wnt3a, epidermal growth factor (EGF), noggin, and R-spondin 1 (WENR). RNA sequencing was used to evaluate whether the cultures maintained segment-specific gene expression patterns by comparing the expression of genes with at least five-fold differential expression between mouse proximal and distal colon tissues to the relative expression of these genes in the monolayer cultures (Table S1).

first: Ying

last: He


title: Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development

city: Saint Louis

first: Kristen

middle: L

last: Kroll

state: MO

address: 320 McDonnell Sciences/660 S. Euclid Ave.

department: Developmental Biology

institute: Washington University School of Medicine

summary: Human embryonic stem cells (hESCs) were specified as ventral telencephalic neuroectoderm (day 4) and then into medial ganglionic emininence (MGE)-like progenitors (day 15) and were subsequently differentiated into cortical interneuron (cIN)-like cells (day 25-35), by modification of previously published protocols. RNA-seq analysis at days 0, 4, 15, 25, and 35 defined transcriptome signatures for MGE and cIN cell identity. Further integration of these gene expression signatures with ChIP-seq for the NKX2-1 transcription factor in MGE-like progenitors defined NKX2-1 putative direct targets, including genes involved in both MGE specification and in several aspects of later cIN differentiation (migration, synaptic function). Among the NKX2-1 direct targets with MGE and cIN enriched expression was CHD2, a chromatin remodeling protein. Since CHD2 haploinsufficiency can cause epilepsy and/or autism, which can involve altered cIN development or function, we evaluated CHD2 requirements in these processes. Transcriptome changes were evaluated in CHD2 knockdown MGE-like progenitors at day 15, revealing diminished expression of genes involved in MGE specification and cIN differentiation including channel and synaptic genes implicated in epilepsy, while later cIN electrophysiological properties were also altered. We defined some shared cis-regulatory elements bound by both NKX2-1 and CHD2 and characterized their ability to cooperatively regulate cIN gene transcription through these elements. We used these data to construct regulatory networks underlying MGE specification and cIN differentiation and to define requirements for CHD2 and its ability to cofunction with NKX2-1 in this process.

overall_design: To comprehensively define changes in gene expression profiles that accompany cortical interneuron (cIN) specification and differentiation process, we have performed RNA sequencing analysis at days 0 (hESCs), 4, 15, 25, and 35. To understand the gene regulatory networks through which NKX2-1 may directly control these processes, we defined its direct targets by performing NKX2-1 ChIP-seq in day 15 MGE-like cells.  Chromatin enrichment for NKX2-1 binding was compared to input and IgG controls. To define the CHD2-dependent gene expression programs during cIN specification, we used CHD2 knockdown (KD) to conduct RNA-seq analysis in d15 CHD2 KD MGE-like cells.

first: Kesavan

last: Meganathan

first: Emily

middle: M

last: Lewis

first: Paul

last: Gontarz

first: Shaopeng

last: Liu

first: Ed

middle: G

last: Stanley

first: Andrew

last: Elefanty

first: James

last: Huettner

first: Bo

last: Zhang


title: PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance [ChIP-seq batch1]

city: Ann Arbor

first: Richard

last: McEachin

state: United States

address: 2800 Plymouth Road

department: DCM&B

institute: University of Michigan

summary: Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA) making developmental regulators therapeutically attractive. Here, we demonstrate diverse functions for PDX1, a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of PanIN-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing EMT and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression.

overall_design: Examination of PDX1 binding in the pancreatic cancer cell line NB490

first: McEachin

last: Richard

first: Nilotpal

last: Roy

first: Kenneth

middle: K

last: Takeuchi

first: Jeanine

last: Ruggeri

first: Peter

last: Bailey

first: David

last: Chang

first: Joey

first: Laura

last: Leonhardt

first: Sapna

last: Puri

first: Megan

middle: T

last: Hoffman

first: Shan

last: Gao

first: Christopher

middle: J

last: Halbrook

first: Yan

last: Song

first: Mats

last: Ljungman

first: Shivani

last: Malik

middle: V

last: Wright

last: Dawson

last: Biankin

first: Matthias

last: Hebrok

first: Howard

last: Crawford


title: BRCA1 Regulates Carbohydrate Metabolism Through its RING Domain and Transcription Factor Oct1

city: Salt Lake City

first: Dean

last: Tantin

state: UT

address: 15 North Medical Drive East

department: Department of Pathology

institute: University of Utah

summary: The tumor suppressor BRCA1 regulates DNA damage responses and multiple other processes. Among these, BRCA1 heterodimerizes with BARD1 to ubiquitylate targets via its N-terminal RING domain. Here we show that BRCA1 promotes oxidative metabolism via degradation of Oct1, a transcription factor with pro-glycolytic/tumorigenic effects. BRCA1 E3 ubiquitin ligase mutation skews cells towards a glycolytic metabolic profile while elevating Oct1 protein. CRISPR-mediated Oct1 deletion reverts the glycolytic phenotype. RNAseq confirms the deregulation of metabolic genes. BRCA1 mediates direct Oct1 ubiquitylation and degradation, and mutation of two ubiquitylated Oct1 lysines insulates the protein against BRCA1-mediated destabilization. Oct1 deletion in MCF-7 breast cancer cells does not perturb growth in standard culture, but inhibits growth in soft agar and xenografts. Oct1 protein levels correlate positively with tumor aggressiveness, and inversely with BRCA1, in primary breast cancer samples. These results identify BRCA1 as an Oct1 ubiquitin ligase that catalyzes Oct1 degradation to promote oxidative metabolism.

overall_design: mRNA profiles of BRCA1-I26A mutant MEFs treated with control CRISPR lentiviral vector, or an Oct1-specific CRISPR construct

first: Karina

last: Vázquez-Arreguín


title: Massively-parallel functional characterization of MAPK1 missense mutants

city: Cambridge

first: Ofir

last: Cohen

state: Massachusetts

address: 415 Main St, Cambridge, MA 02142

department: Cancer Program

institute: Broad Institute

summary: Genome-directed oncology has the potential to revolutionize patient treatment, but is limited by an abundance of rare, uncharacterized and therapeutically uninformative somatic variants. To accelerate characterization of the “long tail” of rare somatic variants, we quantified the activity and drug responsiveness of virtually all possible (99.84%) missense variants in the Ser/Thr kinase MAPK1/ERK2. We identified recurrent and rare hypermorphic and loss-of-function alleles, revealing that variant activity is uncorrelated with mutational frequency. Somatic ERK2 variants displayed variable responses to RAF-, MEK- and ERK-directed therapies, potentially informing clinical treatment strategies for patients whose tumors harbor these alterations. A subset of recurrent and rare somatic variants co-localized on ERK2 protein-protein interfaces, yet engendered contrasting phenotypes based on their specific sub-domain localization. The approach presented here represents an allele-characterization framework that compliments existing computational efforts and supports current and future somatic variant discovery efforts, advancing the promise of genome-guided treatment strategies.

overall_design: Examination of the transcriptional output (mRNA)  for a set of ERK2 Alleles under both trametinib drug treatment and DMSO controls. All experimental conditions were done in duplicates and sequenced in quadruplicates. Thus, each biological sample (48 total) was sequenced in 4 different lanes, resulting in total of 192 sequenced samples

first: Lisa

last: Brenan

first: Davide

last: Cacchiarelli

first: Cory

last: Johannessen


title: Comparison of low temperature transcriptomes between tilapia and zebrafish

city: Shanghai

first: Peng

last: Hu

address: Huchenghuan Road 999

institute: Shanghai Ocean University

summary: Changes in gene expression are thought to be required for maintaining tissue's proper function under cold stress, but little is known about the role of each tissue in setting the cold tolerance ability in fish. Oreochromis niloticus and Danio rerio are two tropical fishes; they differ in their abilities of cold tolerance. In this study, both fishes were exposed to graded cold temperatures, ranging from 28°C to 18°C and 10°C, and the transcriptomes of 8 tissues at each tempeature were sequenced and compared. By characterizing tissue-based gene expression variation between the two fishes we identified some key tissue specific biological processes, which provide a better understating of the typical biological functions of tissues in physiological fitness under cold stress.

overall_design:
Examination of gene expression of 21 tilapia samples (seven tissues at three temperatures)
This submission represents 'tilapia' transcriptome component of study.

first: Liangbiao

last: Chen


title: Comprehensive DNA methylation analysis of the Aedes aegypti genome

city: Heidelberg

first: Cassandra

last: Falckenhayn

address: Im Neuenheimer Feld 580

department: Epigenetics

country: Germany

institute: German Cancer Research Center (DKFZ)

type: Methylation profiling by high throughput sequencing

summary: Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play important roles in virus control and it has been suggested that Dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the A. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the A. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus control.

overall_design: Whole methylome analysis of whole adult A. aegypti mosquitoes mixed female and male (1:1).

first: F

last: Lyko

first: C


title: Identification of a conserved maternal-specific repressive domain in Zelda using Cas9-mediated mutagenesis

city: Madison

first: Melissa

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

summary: In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome only becomes transcriptionally active hours later. Transcriptional activation is tightly coordinated with the degradation of maternally provided mRNAs during this maternal-to-zygotic transition (MZT). In Drosophila melanogaster, the transcription factor Zelda plays an essential role in widespread activation of the zygotic genome. While Zelda expression is required both maternally and zygotically, the mechanisms by which it functions both during and after the MZT remain unclear.  Zelda is a large protein with six C2H2 zinc fingers, but no additional identified domains or predicted enzymatic activities. Using Cas9-mediated genome editing to generate targeted mutations in Zelda, we determined the functional relevance of protein domains conserved amongst Zelda orthologs. Generating these mutations in vivo revealed that neither a conserved N-terminal zinc finger nor an acidic patch were required for activity. Similarly, using a variety of Cas9-enabled approaches we showed that a previously identified splice isoform of zelda is dispensable for viability, and the predicted protein product is not expressed at detectable levels. By contrast, we identified a highly conserved zinc-finger domain that is essential for the maternal, but not zygotic functions of Zelda. A mutation in this zinc-finger domain does not interfere with the capacity of Zelda to activate transcription, but rather leads to a hyperactive form of the protein and enhanced Zelda-dependent gene expression. Embryos inheriting this allele from their mothers die late in embryogenesis. These data define a protein domain critical for controlling Zelda activity and, for the first time, identify a separation between the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are essential for establishing the developmental program during the MZT and that failure to precisely execute this program leads to embryonic lethality.

overall_design: Examination of gene expression profiles from stage 5 wild type embryos or embryos that received maternally deposited zld with a mutation in the second zinc finger (ZnF2).  Three replicates of each were analyzed.

first: Danielle

last: Hamm

first: Elizabeth

last: Larson

first: Markus

last: Nevil

first: Kelsey

last: Marshall

first: Eliana

middle: R

last: Bondra


title: Alteration of treatment of the ALK inhibitor in ALK-amplified neuroblastoma

city: chuo-ku, Tokyo

first: Makoto

last: Miyazaki

state: State...

address: 5-1-1, Tsukiji

country: Japan

institute: National Cancer Research Center Institute

summary:
Anaplastic Lymphoma Kinase (ALK) most frequently mutated in neuroblastoma (NB) and is atractive molecular target for therapy. However, efficacy of the ALK inhibitor against ALK-amplified NB is unclear.
To elucidate genetic alterations induced by treatment of the ALK inhibitor, we compared expression profile between ALK inhibitor-treated and DMSO-treated NB39nu cells using Agilent SurePrint G3 Human GE 8x60K V2 Microarray Kit

overall_design: The ALK inhibitor or DMSO-treated NB39nu cells (parental and resistant), which harbor ALK amplification, were for RNA extraction and hybridization on Agilent microarrays.

first: Yuko

last: Hibiya

first: Masato

last: Enari


title: Transcriptome analysis of Chlamydomonas reinhardtii chloroplast and mitochondria

city: Berkeley

first: Sean

middle: D.

last: Gallaher

state: California

address: 176 Stanley Hall, room 408

department: California Institute for Quantitative Biosciences

institute: UC Berkeley

summary: Here, we report on the transcriptome of the organelles of the micro-alga, Chlamydomonas reinhardtii, sampled under a number of different conditions. The preparation of the RNA-Seq libraries and their analysis were performed using protocols optimized for organellar transcripts. Samples include growth in media +/– Fe, growth in media +/– Cu, diurnal growth samples collected in dark and light, and the sexual cycle.

overall_design: RNA-Seq libraries were prepared from Chlamydomonas reinhardtii sampled under 12 different conditions including: diurnal growth in light, diurnal growth in dark, +Fe, –Fe, +Cu, –Cu, mt+ vegetative, mt– vegetative, mt+ gamete, mt– gamete, zygote, and post-germination.

first: Daniela

last: Strenkert


title: Rhesus Monkey placenta RNA-seq data

city: Bethesda

last: Yang

state: Maryland

address: 6 Center Drive 6B-211

department: DDB

institute: NICHD/NIH

summary: Rhesus Monkey placenta RNA-seq data, the tdf files were generated by using rheMac3 from UCSC.

overall_design: Rhesus Monkey placenta RNA-seq data. Illumina Hiseq2000 were used for sequencing.

first: Gernot

last: Wolf


title: Genome-Wide DNA Methylation Profiling Identifies Differential Methylation in Uninvolved Psoriatic Epidermis

status: Public on Nov 25 2017

city: Linkoping

first: Charlotta

last: Enerbäck

state: Sweden

address: Medical faculty

country: Sweden

institute: Linkoping University

summary: The aim of the study was to investigate the differential methylation in psoriatic lesional epidermis compared to non-lesional or healthy epidermis.

overall_design: Bisulphite converted DNA from 12 samples were hybridised to Illumina Hiseq 1500 system

first: Deepti

last: Verma


title: The effects of mitochondrial- and histone stress on the C. elegans transcriptome

city: Lausanne

first: Maroun

last: Bou Sleiman

state: Vaud

address: Rue Cantonale

department: IBI

country: Switzerland

institute: EPFL

summary: The aim of this study was to explore whether mitochondrial- and histone stress cause any common changes in global gene expression. For this experiment, we performed RNA sequencing from control RNAi, cco-1 RNAi (mitochondrial stress) and his-3 RNAi (histone stress) treated C. elegans. Nematodes were collected at L4 larval stage, and total RNA was extracted with TriPure Isolation Reagent (Roche). An Illumina Truseq stranded mRNA library was prepared for nine samples (three biological replicates for each sample). Single-end sequencing with 100 cycles was performed by running the nine samples on one lane of an Illumina Hiseq 2500, yielding a minimum of 26 million reads per sample. Although the two stressors were found to have divergent effects on global gene expression, some biological processes such as innate immunity, heat shock response and chromatin remodeler expression are commonly upregulated upon both mitochondrial- and histone stress.

overall_design: Wild-type C. elegans was treated with control RNAi, cco-1 RNAi and his-3 RNAi, and gene expression was examined with RNA sequencing

first: Olli

last: Matilainen

middle: S

first: Pedro

last: Quiros

first: Susana

last: Garcia

first: Johan

last: Auwerx


title: RNAseq analysis of NR0B1 LOF

city: La Jolla

first: Max

last: Chang

address: 9500 Gilman Drive

institute: University of California, San Diego

summary: We report analysis of transcriptional chanages following RNAi depletion of NR0B1 or NRF2 depletion in NSCLC cell lines as determined by RNAseq. Additionally we also determine the genomic location of NR0B1 in NSCLC lines by Chip-Seq.

overall_design: Evaulation of RNAseq following NR0B1 depeltion in one cell line and NRF2 depletion in two cell lines.

first: Benjamin

middle: F

last: Cravatt

first: L

last: Bar-Peled


title: Sex differences in peripheral not central immune responses to pain-inducing injury

city: London

first: Franziska

last: Denk

address: Guy's Campus

department: Wolfson SPaRC

country: United Kingdom

institute: King's College London

summary: Women suffer chronic pain more frequently than men. It is not clear whether this is due to differences in higher level cognitive processes or basic nociceptive responses. This study used a mouse model to dissociate these factors and found no differences in peripheral afferent neurons or in the spinal cord immune response to neuropathic injury. However, it did identify potential sexual dimorphisms in peripheral adaptive immune responses.

overall_design: RNA-seq of naïve FACS-purified DRG neurons and MACS-purified DRG neurons after partial sciatic nerve ligation (day 8): comparison of male versus female samples

first: D

last: Lopes

first: S

middle: B

last: McMahon

first: F


title: Genome-wide profiling of gene expression and DNA methylation provides insight into low-altitude acclimation in Tibetan pigs [DNA methylation]

city: Chengdu

first: jin

last: long

address: Hui ming

institute: Sichuan Agricultural University

summary: To explore the exceptional mechanisms of gene expression and DNA methylation that are induced by low altitude environments in Tibetan pigs, we performed a comparative transcriptomic analysis of skeletal muscle in indigenous Tibetan pigs that reside in high altitude regions (～4,000 m) and their counterparts that migrated to the geographically neighboring low-altitude regions (～500 m) for nearly ten generations. We identified protein coding genes that related to hypoxia response (EGLN3 and FLT1), oxygen transport and energy metabolism (TFB2M), and two long non-coding RNAs (TCONS_00039686 and TCONS_00084992) that associated with the regulation of transcription and various nucleolus and organelle lumen, were differentially expressed between Tibetan pigs and their counterparts in low-altitude regions, thus might be the potential candidate regulators in skeletal muscle of low-altitude acclimation in Tibetan pigs. We also found genes embedded in differentially methylated regions between Tibetan pigs and their counterparts in low-altitude regions were mainly involved in ‘Starch and sucrose metabolism’, ‘glucuronosyltransferase activity’ processes, hypoxia and energy metabolism. We envision that this study will serve as a valuable resource for mammal acclimatization research and agricultural food industry.

overall_design: A total of six healthy female Tibetan pigs were used in this study: three free-ranging individuals in Tibetan highland with the average altitude of 4,000 m (high-altitude); three counterparts under experimental conditions with normal daily feeding in geographically neighboring low-altitude regions (Chengdu plain) with average altitude of 500 m (low-altitude), these pigs were descendants of Tibetan pigs that have been raised in lowland for nearly ten generations.

first: Long

last: Jin


title: Single-cell profiling of tumor infiltrating T cells

status: Public on Nov 26 2017

city: Boston

first: Ruben

last: Dries

address: 360 Longwood Ave

department: Computational Biology

institute: DFCI

summary: Immune checkpoint blockade has shown tremendous anti-tumor potential in the clinic. However, these therapies are only effective in a subset of patients, so identification of additional immunomodulatory molecules that enhance the anti-tumor activity of these treatments may expand their clinical utility. In particular, identifying small molecules that complement existing immunotherapies has been relatively unexplored, so we performed a small molecule screen to identify compounds that can enhance co-inhibitory molecule blockade, to improve the anti-tumor adaptive immune response. Our unbiased screen identified inhibitors of cyclin-dependent kinase 4 and 6 (CDK4/6), including the FDA-approved palbociclib, as a class of small molecule compounds that exhibited significant immunostimulatory activity in vitro.  In accordance with our in vitro finding of enhanced NFAT signaling, single-cell RNA-sequencing confirmed that in vivo exposure to CDK4/6 inhibitors enhanced NFAT signaling in tumor infiltrating T cells.  Moreover, our results revealed that CDK4/6 inhibition up-regulated activation molecules and down-regulated suppressive molecules in these cells. CDK4/6 inhibition also increased the number of T cells with activated TCR (T cell receptor) signaling, as well as factors that are important for signal transduction downstream of TCR signaling.  In summary, the impact of CDK4/6i on cell cycle progression and T cell proliferation are balanced favorably towards increased T cell recruitment and enhanced effector cell function, mediated in part by activation of the NFAT family of transcription factors. Further, our results demonstrate that CDK4/6i enhances PD-1 blockade through increased T-cell effector function and inhibition of immune suppressive cytokine production.  While prolonged CDK4/6i treatment could be immunosuppressive due to adverse effects on lymphocyte proliferation, properly timed/sequenced CDK4/6i may potentiate the clinical impact of anti-PD-1/PD-L1 antibodies. As palbociclib is FDA-approved and multiple other CDK4/6 inhibitors are in clinical trials, we expect that this hypothesis will undergo rapid testing in humans.

overall_design: Single-cell comparison of control and CDK4/6 inhibitor treated tumor infiltrating T cells

first: Jiehui

last: Deng


title: Saturation mutagenesis reveals manifold determinants of exon definition

city: Bar Harbor

first: Shengdong

last: Ke

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts we conducted saturation mutagenesis of a 51 nt internal exon in a 3-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high throughput genetics, 5560 minigene molecules were assayed for splicing in HEK293 cells. Over 70% of mutations produced substantial (>2X) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements only a single 15 nt stem-loop, showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and  proteins associated with spliceosome formation (U2AF35, U2AF65, U1Aa, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51 nt test exon simultaneously. Surprisingly, such correlations extended to weak relative protein-sequence affinities. These myriad protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

overall_design: In the work described here we have used deep sequencing to examine the splicing phenotypes produced by saturating a model exon with single and double base substitutions at every position We reasoned that variations on the wild type (WT) theme would provide insights into the prevalence and roles of the sequences that reside in a natural exon. We found that over 70% of these mutations produced substantial (>2-fold) effects on splicing, supporting the idea that most of the computationally predicted ESRs do in fact play a role in splicing. The stability of specific secondary structures was also found to correlate with splicing. We found many strong correlations between splicing and the binding of RNA binding proteins (RBPs) to mutant exons in nuclear extracts, implying that most exonic mutations affected exon definition. Finally, we found significant correlations between splicing and reported binding affinity for a large number of human RBPs. The large scale RBP correlations raise the unanticipated possibility that sequences exhibiting relatively weak affinities can nonetheless influence splicing efficiency. We constructed a 3-exon minigene (Ke et al. 2011) comprised of a 51 nt central target exon WT1-5 (exon 5 of the human Wilms’ tumor gene) surrounded by terminal exons and intronic sequences derived from the Chinese hamster dhfr gene. Thousands of DNA exons were synthesized to specification by primer-extension of a custom DNA microarray. Minigene libraries that incorporated these oligomers into a central exon in a 3‑exon minigene were then prepared (Fig. 1A). Key features of the minigene framework were the provision of strong promoter (CMV) and polyadenylation (SV40) sites and the removal of all start codons from the first exon (Arias et al. 2015) to minimize the chance of nonsense mediated decay (NMD), already unlikely due to the modest size of the exon (Maquat 2004). At each exonic position from 2 to 47 each dinucleotide in the WT1-5 exon was changed to every other possible dinucleotide (Fig. 1B). Positions 1 and 49 to 51 were left unmodified so as not to consider mutations affecting the splice site sequences themselves. The resulting mutant library comprised 555 mutations: 414 double base substitutions (DBSs) and 141 (SBSs) at each position from 2 to 47 and 3 SBSs mutations at position 48. Including the wild type (WT), the total number of distinct molecules numbered 556 (Fig. 1B). Splicing of the WT exon takes place with an efficiency (percent spliced in, psi; presented here as a proportion) of 0.065 (Ke et al. 2011); this low level is the result of depriving WT1‑5 of its natural flanking intronic sequences (Zhang et al. 2005c). The modest splicing efficiency of the WT minigene was purposely engineered to allow detection of mutations that increase (up to 16X) as well as decrease (to 0) splicing efficiency.

first: Lawrence

last: Chasin


title: WhiB6 regulation of ESX-1 gene expression is controlled by a negative feedback loop in Mycobacterium marinum

city: East Lansing

first: Robert

middle: B.

last: Abramovitch

state: MI

address: 567 Wilson Road

department: Microbiology and Molecular Genetics

institute: Michigan State University

summary: WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the whiB6 gene and genes encoding ESX-1 substrates were reduced.

overall_design: Comparison of M. marinum transcriptional profiles in WT M. marinum, an ochre mutant in the eccCb1 gene, a whiB6 deletion mutant and complemented strains.

first: Rachel

last: Bosserman

first: Tiffany

last: Nguyen

first: Kevin

last: Sanchez

first: Alexandra

last: Chirakos

first: Micah

last: Ferrell

first: Matthew

last: Champion

first: Patricia


title: miRNA-seq in MKN45 cells after stable knockdown of CUL4B expression

city: Jinan

first: Han

last: Bo

address: #44, Wenhua Xi Rd

institute: Shandong University Medical School

summary: As miRNAs are increasingly shown to be vital regulators of cellular behavior as well as diseases including cancer, it is highly important to identify and characterize miRNAs that are regulated by CUL4B. Here, 106 miRNAs were identified upregulated in MKN45-4BI cells compared with NS cells.

overall_design: Using Illumina HiSeq 2000, we analyzed the miRNA expression levels in MKN45 cells that were stable transfected with contol(NS)or shCUL4B(4BI).

first: Bo

last: Han

first: Mei

last: Qi


title: Genome-wide profiling of gene expression and DNA methylation provides insight into low-altitude acclimation in Tibetan pigs [RNA-seq]

first: jin


title: PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance [ChIP-seq batch3]

city: Ann Arbor

first: Richard

middle: C

last: McEachin

address: 2800 Plymouth Road

institute: University of Michigan

summary: Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA) making developmental regulators therapeutically attractive. Here, we demonstrate diverse functions for PDX1, a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of PanIN-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing EMT and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression.

overall_design: Examination of PDX1 binding in the pancreatic cancer cell line NB490

first: McEachin

last: Richard

first: Nilotpal

last: Roy

first: Kenneth

last: Takeuchi

first: Jeanine

last: Ruggeri

first: Peter

last: Bailey

first: David

last: Chang

first: Joey

last: Li

first: Laura

last: Leonhardt

first: Sapna

last: Puri

first: Megan

last: Hoffman

first: Shan

last: Gao

first: Christopher

last: Halbrook

first: Yan

last: Song

first: Mats

last: Ljungman

first: Shivani

last: Malik

last: Wright

middle: W

last: Dawson

first: Andrew

last: Biankin

first: Matthias

last: Hebrok

first: Howard

last: Crawford


title: PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance [ChIP-seq batch2]

first: Richard

last: McEachin

first: McEachin

last: Richard

first: Nilotpal

last: Roy

last: Takeuchi

last: Ruggeri


title: PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance

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

overall_design: Refer to individual Series


title: Plagl1/Zac1 regulates extracellular matrix genes and the imprinted gene network

status: Public on Nov 24 2017

city: Montpellier

first: Tristan

last: Bouschet

address: 141 rue de la cardonille

department: CNRS 5203, INSERM U661

institute: Institut de Génomique Fonctionnelle

summary: In order to determine the targets of the imprinted transcription factor Zac1, we compared wild type and Zac1-mutant MEFs and measured the differentially expressed genes by RNA-seq. Genes deregulated in the absence of Zac1 include genes belonging to the Imprinted Gene Network and genes coding for the extra-cellular matrix.

overall_design: Two biological replicates of MEFs wildtype and Zac1-mutant were cultivated at different time point corresponding to proliferative and quiescent states. Genes differentially expressed between WT and Zac1-mutant MEFs were determined using RNA-seq.

first: Annie

last: Varrault

first: Christelle

last: Dantec

first: Anne

last: LeDigarcher

first: Laëtitia

last: Chottard

first: Benoit

last: Bilanges

first: Hugues

last: Parrinello

first: Emeric

last: Dubois

first: Stéphanie

last: Rialle

first: Dany

last: Séverac

first: Laurent

last: Journot


title: Serotype-specific gene expression in GAS

city: Reno

first: Paul

last: Sumby

state: NV

address: 1554 N. Virginia St.

department: Microbiology & Immunology

institute: University of Nevada

summary: We used RNA-Seq to compare gene expression between representative serotype M1 and M3 GAS isolates

sample_organism:
Streptococcus pyogenes serotype M1
Streptococcus pyogenes serotype M3

overall_design: Triplicate THY broth cultures of MGAS2221 (M1) and MGAS10870 (M3) were growth to the mid-exponential phase of growth.  RNA was isolated and used in RNA-Seq analysis.


title: PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance [RNA-seq]

last: McEachin

overall_design: Examination of the effects of PDX1 binding on gene expression in the pancreatic cancer cell line NB490.  We evaluated the effect of PDX1 binding by comparing gene expression

first: Kenneth

first: Jeanine

first: Peter

last: Bailey

first: David

last: Chang

first: Joey

last: Li

first: Laura

last: Leonhardt

first: Sapna

last: Puri

first: Megan

last: Hoffman

first: Shan

last: Gao

first: Christopher

last: Halbrook

first: Yan

last: Song

first: Mats

last: Ljungman

first: Shivani

last: Malik

last: Wright

last: Dawson

first: Andrew

last: Biankin

first: Matthias

last: Hebrok

last: Crawford


title: Genome-wide profiling of somato-dendritic mRNA distribution in the hippocampus in RNG105/caprin1 conditional knockout (cKO) mice

city: Okazaki

first: Rie

last: Ohashi

address: Nishigonaka 38, Myodaiji

institute: National Institute for Basic Biology

summary:
Background: mRNA localization and translation in neuronal dendrites are the basis for long-term synaptic plasticity and memory formation. RNA granules, membrane-less macromolecular assemblies in which dendritic mRNAs are recruited, are thought to contribute to these processes. RNG105/caprin1 is an RNA granule assembly factor involved in mRNA transport.
Results: Genome-wide profiling of mRNA distribution in distinct hippocampal layers revealed dendritically and somatically enriched mRNAs, and demonstrated marked reduction in the differential somato-dendritic localization of the mRNAs in RNG105 cKO mice.
Conclusion: RNG105 is an essential factor for the localization of many, but particularly for specific mRNAs localized to dendrites.

overall_design: mRNAs isolated from stratum pyramidale (SP) and stratum radiatum (SR) in the CA1 region of the hippocampus from control mice and RNG105 cKO mice were deep sequenced in triplicate using a HiSeq1500 (Illumina).

first: Shuji

last: Shigenobu

first: Nobuyuki

last: Shiina


title: The pluripotency factor Zfp281 coordinates transcriptional and epigenetic control of epiblast maturation

city: New York

first: Jianlong

last: Wang

state: New York

department: Department of Medicine

institute: Columbia University Irving Medical Center


title: Transcriptome and Functional Analyses Reveal Roles For Regulators of Epigenetic States, Micro RNA Processing, And Long Non-Coding RNA In Myocyte Dedifferentiation: Insights Into Reprogramming A “Post-Mitotic” Cell

first: Alon

last: Kahana

address: 1000 Wall St.

department: Ophthalmology and Visual Sciences

summary:
Purpose: The ability of adult zebrafish tissues to undergo dedifferentiation provides an opportunity to probe the molecular underpinnings of cell identity and reprogramming. Zebafish muscle regeneration utilizes dedifferentiation to reprogram mature multinucleated myocytes into dedifferentiated myoblast that re-enter the cell cycle. A unique advantage of this system is that the regenerating cell mass is large and fairly homogenous, facilitating genomics approaches to uncovering the underlying biology.
Methods: To better understand cellular reprogramming of mature myocytes, we temporally analyzed the changing transcriptome leading up to the proliferative switch. RNA was obtained after Laser Micro-dissection (LMD) of Control, 9 hour post-injury (HPI) or 18 HPI using Trizol and micro column purification. Illumina's TruSeq Stranded mRNA Library Prep Kit and 0.1 - 4 µg total mRNA from pooled purified RNA samples were used for performing ribosomal-depletion (Ribo-Zero Gold rRNA Removal Kit, Illumina) and library preparation. Sequencing was performed by the UM DNA Sequencing Core, using an Illumina Hi-Seq 2000 (50-cycle, single end read) platform.
Results: Clustering and functional annotation of differentially expressed genes highlighted the importance of catabolic and phagocytic processes upregulation at 9 and 18 hours post injury (hpi). Furthermore, genes encoding principle regulators of chromatin states were actively re-regulated during the reprogramming process. Utilizing the accessibility of these tissues in the zebrafish model, kKnockdown experiments enabled in vivo validation and phenotypic analysis of candidate genes and pathways for their roles in genomic and cellular reprogramming. Additionally, we found that despite of their low expression levels, lncRNAs were highly represented in gene clusters with dynamic, “switch-like” expression profiles, and that miRNA processing was also found important for reprogramming
Conclusions:  We conclude that reprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires both heritable yet nuanced epigenetic alterations and molecular switches that involve transcription factors, miRNA and lncRNA, while maintaining the lineage restriction of the cell of origin.

overall_design: Early time points post injury (9 & 18 hours) mRNA and lncRNA profiles of Zebrafish lateral eye muscle (EOM) were generated by deep sequencing, in quadruplicate, using Illumina Hi-seq.

first: Alfonso

last: Saera-Vila

first: Phillip

last: Kish


title: RNA-seq analysis of mouse brain endothelial cells and glioma endothelial cells

city: DALLAS

first: KANGSAN

last: KIM

state: TX

address: 5323 Harry Hines Blvd

department: Pathology

institute: UT Southwestern Medical Center

summary: Glioblastoma multiforme (GBM) is a highly aggressive and vascularized malignant brain tumor. Anti-VEGF therapy is widely used for the treatment of GBM, however it has shown only minor impact on patient survival. Thus, more precise molecular mechanisms for glioma angiogenesis are needed for the advance in the treatment of GBM. Here, we investigated gene expression profile of brain endothelial cells and glioma endothelial cells using RNA sequencing to validated special molecular features of glioma endothelial cells.

overall_design: To reveal molecular mechanisms underlying vascular characteristics of high-grade glioma, we established mouse orthotopic glioma model by intracranially injecting GL26l glioma cells into congenic B6 genetic background mice. We further compared tumor vessels with non-tumor brain vessels at the molecular level. For in-depth understanding of different gene expression between tumor vessels and brain vessels, we purified endothelial population from tumor and brain tissues and performed RNA-sequencing analysis. Three independent sets of RNA were obtained from normal brain endothelial cells and glioma endothelial cells.

first: Kangsan

last: Kim

first: Il-Kug

first: Injune


title: Defined IFNg levels improve anti-tumor and preclinical testing of immunotherapy in hosts with cancer and underlying autoimmunity

city: Frederick

first: Jack

last: Chen

state: MD

address: SAIC-Frederick Miller Dr

institute: NIH

summary: Most immunotherapies benefit only subsets of cancer patients, except those with autoimmune diseases due to dysregulation of their immune system. Here, the ARE-del mouse model featuring IFNg-autoimmunity allowed us to examine the relationship between cancer, autoimmunity and immunotherapy. We demonstrate that systemic levels of IFNg below 20 pg/ml in ARE-del+/- (HET) mice exerts moderate anti-tumor effects and increase (p=0.02; ANOVA) overall survival (OS); while higher levels did not that replicate in vivo the anti-tumor “bell shape” response of IFNg. Having confirmed that PD1 and CD40 are expressed in tumor-bearing ARE-del mice, anti-PD1 and anti-CD40 alone or combined were used to improve the antitumor effects of IFNg in ARE-del+/- (HET) mice. In ARE-del+/+ (WT) mice, PD1/CD40 inhibited tumor growth and improve OS because it prevented increased infiltration and trapping of tumor associated macrophages (TAMs) into necrotic areas, an event also decreased by anti-CD40 but promoted by anti-PD1. Conversely, in ARE-del+/- (HET) mice, MHC class II expressing TAM infiltration associated with severe hematological and liver adverse effects hampered the efficiency of PD1/CD40, despite significant tumor growth inhibition and strong induction of IFNg signaling. Thus, excessive activation of IFN negatively impacted immune outcome. Given the lack of consensus on the right levels of IFNg, our data show the upper limit of IFNg that define the efficacy of checkpoint inhibitors in tumor-bearing hosts with autoimmune disease.

overall_design: Subcutaneous B16F10 tumor mRNA profiles from wild type (WT) and ARE-del heterozigous female mice treated with anti-PD1, anti-CD40 or anti-PD1/CD40 mAB were generated by deep RNA sequencing.

first: Julio

last: Valencia

first: Howard

last: Young


title: Genome-wide profiling of gene expression and DNA methylation provides insight into low-altitude acclimation in Tibetan pigs

first: jin

last: long


title: In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation.

city: Seattle

first: James

middle: William

last: MacDonald

state: WA

address: 4225 Roosevelt Way NE

department: Environmental and Occupational Health Sciences

institute: University of Washington

summary: In utero exposure to diesel exhaust particulates has been associated with increased adult susceptibility to heart failure in mice but the mechanisms by which this exposure promotes susceptibility are poorly understood. To identify potential transcriptional effects that mediate this susceptibility, we have performed RNA-seq analysis on adult hearts from mice exposed to diesel exhaust in utero and that have subsequently undergone transverse aortic constriction. We identified three target genes, Mir133a-2, Ptprf and Pamr1, which demonstrate dysregulation after exposure and aortic constriction. Examination of expression patterns in human heart tissue indicate a correlation between expression and heart failure. We subsequently examined for DNA methylation modifications at these candidate loci in neonatal cultured cardiac myocytes after in utero exposure to diesel exhaust and found that the promoter for Mir133a-2 is differentially methylated.

overall_design: Female mice were paired with male mice for timed mating in filtered air (FA). After observation of a vaginal plug, pregnant mice were put into FA or diesel exhaust (DE) with exposures beginning at E0.5 and lasting until E17.5. Neonates were used for neonatal cardiomyocyte preparation or saved for adult surgery. Offspring were raised in FA until 11 weeks, at which time they underwent baseline echocardiography and transverse aortic constriction at 12 weeks using a 27-gauge needle. Hearts from these mice were harvested at 15 weeks, rapidly frozen in liquid nitrogen and stored at -80C prior to RNA isolation.

first: Jamie

last: Goodson

first: Chad

last: Weldy

first: Yonggang

last: Liu

first: Theo

last: Bammler

first: Wei-Ming

last: Chien

first: Michael

last: Chin


title: Effects of RPAP1 depletion on mRNA gene expression (RNA-seq) and RNA Pol II abundance (ChIP-seq) in primary mouse embryonic fibroblasts or embryonic stem cells.

city: Madrid

first: Cian

last: Lynch

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Molecular Oncology

country: Spain

institute: Spanish National Cancer Research Centre (CNIO)

summary: Here, we investigated the role of RPAP1 in mammalian cell identity.  As in plants, interfering with RPAP1 function did not affect the viability of mouse embryonic stem cells, but severely impaired their differentiation capacity (RNA-Seq#2 data, below).  Conversely, in somatic cells, RPAP1 was essential for the expression of lineage specifying factors and viability (RNA-seq#1 data, below).  Moreover, inhibition of RPAP1 triggers dedifferentiation and facilitates reprogramming into pluripotent stem cells.  Mechanistically, RPAP1 maintains transcribing Pol II levels and Pol II Ser5 phosphorylation, particularly on developmental genes (ChIP-seq data, below).

overall_design:
Series includes two RNA-seq datasets, and one ChIP-seq dataset:

In the RNA-seq#1 data (below), three independent lines of mouse embryonic fibroblasts (#1, #3, #5) were lentivirally infected with shRNAs against RPAP1 (shRP) or a non-targeting control (SCR) followed by puromycin selection.  To assess differential mRNA expression, RNA-seq was performed at day 3 after lenti-shRNA delivery, comparing control (SCR) versus RPAP1-depleted cells (shRP).

In the RNA-seq#2 data (below), three independent replicates (A, B, C) of mouse ES cells were lentivirally infected with shRNAs against RPAP1 (shRP) or a non-targeting control (SCR) followed by puromycin selection.  The ES cells were then induced to differentiate by LIF removal for 24 hrs, and to assess differential mRNA expression, RNA-seq was performed, comparing control (SCR) versus RPAP1-depleted cells (shRP).

In the ChIP-seq data (below), three independent lines of mouse embryonic fibroblasts were lentivirally infected with shRNAs against RPAP1 (shRP) or a non-targeting control (SCR) followed by puromycin selection.  To assess any changes in RNA Pol II abundance on genes, ChIP-seq for total Pol II, or Ser5P Pol II, was performed at day 3 after lenti-shRNA delivery, comparing control (SCR) versus RPAP1-depleted cells (shRP).

first: Osvaldo

last: Graña


title: Genome-wide characterization of Zac1 target genes reveals its role as a broad regulator of the imprinted gene network including extracellular matrix genes

first: Tristan

last: Bouschet


title: FTY720 (fingolimod) regulates key target genes essential for inflammation in microglial cells

city: Ansan

first: Young Gyu

last: Chai

state: Gyeonggi-do

address: 55 Hanyangdaehak-ro

department: Molecualr & Life Science

country: South Korea

institute: Hanyang Univ.

summary: Although microglial have an essential role in host defense in the brain, the abnormal activation of microglia can lead to devastating outcomes, such as neuroinflammation and neurodegeneration. Emerging evidence increasingly supports that FTY720 (fingolimod), an FDA-approved drug has beneficial effects in the CNS  on brain cells and more recently immunosuppressive activities in microglia via modulation of the S1P1 receptor. However, the exact molecular aspects of FTY720 contribution in microglia remain largely unaddressed. To understand the molecular mechanisms underlying the roles of FTY720 in microglia, we performed gene expression profiling in resting, FTY720, LPS and LPS+FTY720 challenged primary microglial cells (PM) isolated from 3-day-old ICR mice and we identified FTY720 target genes and co-regulated modules that were critical in inflammation. By examining RNA-sequencing and binding motif datasets from FTY720 suppressed LPS-induced inflammatory mediators, we also identified unexpected relationships between the inducible transcription factors (TFs), motif strength, and transcription of key inflammatory mediators. Furthermore, we show that FTY720 controls important inflammatory genes targets by modulating STAT1, and IRF8 level at their promoter site. Our unprecedented findings demonstrate that FTY720 could be a useful therapeutic application for neuroinflammatory diseases associated with microglia activation as well as provide a rich resource and framework for future analyses of FTY720 on microglia interaction.

overall_design: Murine microglial PM, were treated with FTY720 (1 uM), and with LPS (10 ng/ml) for 4 hrs.


title: Zac1 is a regulator of the imprinted gene network (RNA-seq)

summary: In order to determine the imprinted transcription factor Zac1 targets, we overexpressed Zac1 in a mouse insulinoma cell line and measured  the regulated expressed genes by RNA-seq. We have shown that Zac1 regulates many genes belonging to the Imprinted Gene Network, including genes coding for the extra-cellular matrix.

overall_design: Determination of Zac1 target genes in transfected Min6 cells (4 biological replicates) using RNA-seq, .

first: Annie

last: Varrault

first: Christelle

last: Dantec

first: Anne

last: LeDigarcher

first: Laëtitia

last: Chottard

first: Hugues

last: Parrinello

first: Emeric

last: Dubois

first: Laurent

last: Journot


title: Transcriptomic hallmarks of bone remodeling revealed by RNA-seq profiling in blood of Arabian horses during racing training regime

city: Balice

first: Kacper

last: Zukowski

address: Krakowska 1 Street

country: Poland

institute: National Research Institute of Animal Production

summary:
Purpose: RNA-seq method was used to identify differentially expressed genes in whole blood involved in bone remodelling during racing training in young Arabian horses.
Methods: The comparisons of transcriptomes of whole blood between GI (6 untrained horses) and GII (4 horses after 24 weeks of flat racing training) has been performed. The RNA was isolated using MagMAX™-96 Total RNA Isolation Kit and 400ng were directed to cDNA libraries construction Illumina deep sequencing (75 single-end cycles on Illumina HiScan SQ platform). The bioinformatics analysis include the RSEM and STAR aligner. The raw reads were aligned to the Equus caballus reference genome. Differentially expressed genes were detected by DESeq2.The validation of RNA-seq results were performed by qPCR.
Results: After comparison of whole blood transcriptomes from control and trained horses, we identified 1290 training induced genes. Among significant deregulated molecules we recognized twelve genes potentially involved in metabolism of bone (BGLAP, CTSK, TYROBP, PDLIM7, SLC9B2, TWSG1, NOTCH2, IL6ST, VAV3, NFATC1, CLEC5A, TXLNG). Within significantly deregulated pathways one from the most overrepresented was associated with osteoclast differentiation.
Conclusions: In the presented study, we identified a panel of DEGs which should be evaluated as candidate biomarkers for bone homeostasis indicators in Arabians performed racetrack. In our results, we pinpointed that intense training has itself effect on immature skeletal system. Thus, further studies are essential to establish biomarkers which could be used in assessment of bone remodelling state during training for race track competition.

overall_design: The samples of whole blood from 10 Arabian horses were sequenced in 75 single-end cycles on Illumina HiScan SQ platform in 6 technical repetitions.

first: Monika

last: Stefaniuk-Szmukier

first: Katarzyna

last: Ropka-Molik

last: Żukowski

last: Piórkowska

last: Bugno-Poniewierska


title: Zac1 is a regulator of the imprinted gene network (ChIP-seq)

summary: In order to determine the imprinted transcription factor Zac1 targets, we overexpressed Zac1 in a neuroblastoma cell line and measured both the regulated expressed genes by Differential Gene Expressed analysis and Zac1 binding sites throughout the mouse genome by ChIP-seq. We have shown that Zac1 regulates and binds closed to many genes belonging to the Imprinted Gene Network.

overall_design: Determination of Zac1 binding sites in transfected Neuro-2A cells (2 biolobical replicates) using ChIP-seq, with the chromatin input as a control.


title: SOX7 and ETS factors regulate an endothelial transcriptional program during vascular development (DamID)

city: Manchester

first: Andrew

middle: Joshua

last: Lilly

address: Wilmslow Road

department: Stem Cell Haematopoiesis

institute: CRUK Manchester Institute

summary: Vascular development involves complex changes in gene expression necessary to dictate the behaviour of differentiating endothelial cells within the emerging vascular network.  However, the transcriptional programs regulating vascular development are poorly understood.  Here we use a DamID approach to profile for the first time the global DNA binding pattern of SOX7, FLI1 and ERG in endothelial precursor cells.  We uncover that SOX7, FLI1 and ERG bind together at endothelial specific regulatory regions to drive a transcriptional program important for vascular development.

overall_design: DamID for SOX7, FLI1 and ERG relative to WT DAM only control

first: Valerie

last: Kouskoff


title: Genome-wide characterization of Zac1 target genes reveals its role as a broad regulator of the imprinted gene network including extracellular matrix genes.

last: Bouschet

overall_design: Determination of the genes regulated after Zac1 overexpression by measuring the transcriptomes of CAT (Chloramphenicol Acetyl Transferase) and Zac1-transfected Neuro-2A cells using Digital Gene Expression. 2 biological (transfection) replicates were perfomed.


title: The conserved transcriptional landscapes in human spermatogenesis

city: Rennes

first: Frédéric

last: Chalmel

state: France

address: 9 avenue du Pr. Léon Bernard

department: Physiology and physiopathology of the urogenital tract

institute: Inserm U1085-Irset

summary: Spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-coding transcriptome, little effort has yet been devoted to the systematic identification of lncRNAs expressed during spermatogenesis in human. We report high-resolution expression profiling of human male germ cells using Illumina next-generation sequencing technology and highly enriched testicular cell populations. 25,161 high-confidence transcripts were reconstructed and classified into eleven expression patterns.  Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource of novel loci expressed during spermatogenesis that significantly contributes to the human genome annotation. A graphical display of the data is conveniently accessible through the ReproGenomics Viewer (RGV) at http://rgv.genouest.org.

overall_design: Genome-wide expression profiling analysis using Illumina next-generation sequencing technology and highly enriched testicular cell populations

first: Antoine

middle: D

last: Rolland


title: DNA methylation state is associated with the formation of loops and links in hematopoietic stem cells [RNA-seq]

city: HOUSTON

first: Jianzhong

last: Su

state: TEXAS

address: 1 Baylor Plaza

institute: Baylor college of Medicine

summary: We present the high resolution HiC study of genome folding in primary hematopoietic stem and progenitor cells. We investigated the relationship of chromosomal contacts with other epigenetic marks, particularly DNA methylation which is known to be important for appropriate stem cell differentiation.

overall_design: We use RNA-Seq to probe relationship of chromosomal contacts and gene expression changes in hematopoietic stem cell

first: Jaime

middle: M

last: Reyes

first: Mira

last: Jeong

first: Margaret

middle: A

last: Goodell

first: Erez

middle: L

last: Aiden


title: Knockout of the Arp2/3 complex in epidermis causes a psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2 [Epidermis ssRNA-Seq]

status: Public on Nov 23 2017

city: Amsterdam

first: Metello

last: Innocenti

address: Plesmanlaan 121

country: Netherlands

institute: NKI

summary: Arp2/3 complex assembles branched actin filaments key to many cellular processes, but its organismal roles remain poorly understood. Here we employed conditional arpc4 knockout mice to study the function of the Arp2/3 complex in the epidermis.We found that depletion of the Arp2/3 complex by knockout of arpc4 results in skin abnormalities at birth that evolve into a severe psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2. Knockout of arpc4 in cultured keratinocytes was sufficient to induce nuclear accumulation of Nrf2, upregulation of Nrf2-target genes and decreased filamentous actin levels. Furthermore, pharmacological inhibition of the Arp2/3 complex unmasked the role of branched actin filaments in Nrf2 regulation. Consistently, we unveiled that Nrf2 associates with the actin cytoskeleton in cells and binds to filamentous actin in vitro Finally, we discovered that Arpc4 is downregulated in both human and mouse psoriatic epidermis. Thus, the Arp2/3 complex affects keratinocytes' shape and transcriptome through an actin-based cell-autonomous mechanism that influences epidermal morphogenesis and homeostasis.

overall_design: Gene expression profile of  wt and ARPC4 ko epidermis

first: Rob

last: van der Kammen


title: Knockout of the Arp2/3 complex in epidermis causes a psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2 [Keratinocytes ssRNA-Seq]

overall_design: Gene expression profile of  wt, ARPC4 ko and EGFP-Nrf2 expressing keratinocytes.


title: Genome wide identification of MCM7 binding sites as replication origins

city: Fukuoka

first: Nozomi

last: Sugimoto

address: 3-1-1, Maidashi, Higashiku

institute: Kyushu University

summary: Here, we precisely determined MCM7 binding sites inHeLa cells by ChIP-Seq, classified them into firing and dormant replication origins using SNS data deposited, and analyzed their association with various chromatin signatures.

overall_design: ChIP-Seq of MCM7 in HeLa cells. BioProject: PRJDB2761, Accession: DRA001271 (DRP001342); BioProject: PRJDB5845, Accession: DRA005864 (DRP003852).

first: Kazumitsu

last: Maehara

first: Kazumasa

last: Yoshida

first: Yasuyuki

last: Ohkawa

first: Masatoshi

last: Fujita


title: Comparative ribosome profiling uncovers a dominant role for translational control in Toxoplasma gondii

city: Edinburgh

first: Musa

last: Hassan

state: Scotland

address: Easter Bush

department: Roslin

institute: University of Edinburgh

summary:
The lytic cycle of the protozoan parasite Toxoplasma gondii, which involves a brief sojourn in the extracellular space, is characterized by defined transcriptional profiles. For an obligate intracellular parasite that is shielded from the cytosolic host immune factors by a parasitophorous vacuole, the brief entry into the extracellular space is likely to exert enormous stress. Due to its role in cellular stress response, we hypothesize that translational control plays an important role in regulating gene expression in Toxoplasma during the lytic cycle. Unlike transcriptional profiles, insights into genome-wide translational profiles of Toxoplasma gondii are lacking.
We have performed genome-wide ribosome profiling, coupled with high throughput RNA sequencing, in intracellular and extracellular Toxoplasma gondii parasites to investigate translational control during the lytic cycle.
Results: Although differences in transcript abundance were mostly mirrored at the translational level, we observed significant differences in the abundance of ribosome footprints between the two parasite stages. Furthermore, our data suggest that mRNA translation in the parasite is potentially regulated by mRNA secondary structure and upstream open reading frames.

overall_design: We compared ribosome footprints in intracellular and extracellular parasites.


title: BRCA1 suppresses microhomology-mediated tandem duplications at Tus/Ter-stalled replication forks

city: Stanford

first: Richard

last: Frock

address: 269 Campus Drive

department: Radiation Oncology

institute: Stanford University

summary: Small ~10 kb microhomology-mediated tandem duplications (“Group 1 TDs”) are abundant in BRCA1-linked but not BRCA2-linked breast cancer genomes. Here, we define the mechanism underlying this rearrangement signature. We show that BRCA1, but not BRCA2, suppresses TDs at a Tus/Ter site-specific chromosomal replication fork barrier in primary mammalian cells. BRCA1 has no equivalent role at chromosomal double strand breaks, indicating specificity for the stalled fork response. Two motor proteins—FANCM and the Bloom’s syndrome helicase—suppress Tus/Ter-induced TDs in BRCA1 mutants, revealing the existence of a multi-gene TD suppressor network. TDs arise by a “replication restart-bypass” mechanism terminated by end joining or microhomology-mediated template switching, the latter forming complex TD breakpoints. We show that solitary DNA ends form directly at Tus/Ter, implicating misrepair of these lesions in TD formation. We find that BRCA1 inactivation is strongly associated with Group 1 TDs in ovarian cancer. The Group 1 TD phenotype may be a general signature of BRCA1-deficient cancer

overall_design: LAM-HTGTS from a Cas9/gRNA bait DSB 30kb away from the 1xGFP Ter array cassette inserted in the ROSA26 locus

first: Nicholas

last: Willis

first: Frederick

last: Alt

first: Ralph

last: Scully


title: DNA methylation state is associated with the formation of loops and links in hematopoietic stem cells [HiC]

first: Jianzhong

last: Su

summary: We present the high resolution HiC study of genome folding in primary cells, specifically hematopoietic stem and progenitor cells. We investigated the relationship of chromosomal contacts with other epigenetic marks, particularly DNA methylation and histone modifications which is known to be important for appropriate stem cell differentiation.

overall_design: We used in situ Hi-C to probe relationship of chromosomal contacts and epigenetic modification.

first: Muhammad

last: Shamim


title: Endothelial cell differentiation is encompassed by changes in long range chromatin interactions between inactive chromatin regions

city: Kuopio

first: Henri

last: Niskanen

department: A.I. Virtanen Institute

country: Finland

institute: University of Eastern Finland

summary: Endothelial cells, which constitute the luminal layer of blood and lymphatic vessels, differentiate from mesodermal progenitors during a process called vasculogenesis. By analyzing changes in chromatin interactions in human umbilical vein endothelial cells (HUVEC) compared to human embryonic stem cells (ESC) and mesendoderm cells (MSC), we identified regions exhibiting endothelial-specific compartmentalization and changes in the degree of connectivity within topologically associated domains (TADs). These EC-specific regions of chromatin interactions were characterized by EC-specific transcription, binding of lineage-determining transcription factors and cohesin. In addition, we developed a method that identified for the first time hundreds of cell-type specific long-range interactions between TADs. Most of these interactions were connections between regions enriched for H3K9me3, suggesting that majority of long-range interactions form between regions of heterochromatin involving pericentromeric regions during cellular differentiation. Secondly, we demonstrate that the hierarchy of chromatin domains is already pre-established in ESCs suggesting that higher level chromatin aggregation is conserved throughout cellular differentiation. Finally, we investigated the effect of hypoxia on transcription and chromatin interactions of ECs. Although hypoxic stress altered the expression of hundreds of genes, it had only a minimal effect on the chromatin compartmentalization, on the amount of local interactions within TADs or long range interactions. Collectively our results show that large structural rearrangements establish chromatin architecture required for functional endothelium and this architecture remains mostly unchanged in response to hypoxic stress.

overall_design: Chromatin interactions of HUVECs were analyzed genome-wide using tethered conformation capture (TCC) in normoxia and hypoxia (8h, 1% O2). Transcriptional response was studied using global run-on sequencing (GRO-seq), cohesin binding using ChIP-seq for RAD21 and histone modifications using ChIP-seq for H3K9me3 and H3K27me3.

first: Minna

middle: U

last: Kaikkonen


title: cis-regulatory logic of Shh expression reveals common history of unpaired and paired fins

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

institute: CABD/CSIC

summary: Despite their evolutionary, developmental, and functional importance the origin of vertebrate paired appendages remains a mystery. Classical ideas center on three hypotheses: that paired fins relate to gill arches, are derived from a fin fold, or that median fins evolved first and paired fins arose by co-option of ancient genetic patterning modules. Importantly, the third hypothesis makes specific predictions about the function and phylogenetic history of cis-regulatory elements (CREs) involved in appendage patterning. Here we show that a major enhancer for Sonic hedgehog (Shh, a key diffusible morphogen essential for proper growth and patterning of limbs and paired fins) is deeply shared between paired and unpaired fins in fish and with the limbs of mice. In mice, a single CRE termed ZRS is solely responsible for coordinating Shh expression in limbs. We use transgenic assays in zebrafish and mouse to trace the functional equivalence of the ZRS across gnathostome phylogeny and its likely absence in agnathans. CRISPR/Cas9-mediated deletion of the medaka core ZRS sequence, inspection of shh epigenetic landscape and in vivo enhancer assays reveal the existence of ZRS shadow enhancers in both teleost and human genomes. Strikingly, a complete deletion of the medaka ZRS, an experiment expected to only affect the paired fins, results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and medial fins, and that paired fins likely emerged by the co‐option of developmental programs established in the median fins of early vertebrates. Later on, this critical ZRS-driven Shh function was reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.

sample_organism:
Oryzias latipes
Mus musculus

platform_organism:
Oryzias latipes
Mus musculus

overall_design: 4C-seq experiments from Shh promoter and ZRS enhancer in mouse and medaka.

first: Joaquín

last: Letelier

first: Elisa

last: de la Calle-Mustienes

first: Joyce

last: Pieretti

first: Silvia

last: Naranjo

first: Ignacio

last: Maeso

first: Tetsuya

last: Nakamura

first: Neil

last: Shubin

first: Igor

last: Schneider

last: Martinez-Morales

first: José

last: Gómez-Skarmeta


title: The pluripotency factor Zfp281 coordinates transcriptional and epigenetic control of epiblast maturation [RNA-seq]

first: Jianlong

last: Wang

summary: The early development of mouse embryos involves a tightly regulated series of lineage specification events. Following implantation, these events include maturation of the pluripotent epiblast and simultaneous establishment of the embryonic axes. Here we report that Zfp281, a transcription factor shown to be required for transitioning cells in culture from naïve to primed states of pluripotency, is essential for mouse embryonic development by regulating epiblast maturation. Transcriptomic analyses of Zfp281 mutant embryos revealed a failure in activating Nodal signaling in the epiblast leading to failure in establishing the anterior-posterior axis, and impaired epiblast maturation. Our study establishes Zfp281 as a critical factor that coordinates transcriptional and epigenetic control of pluripotent epiblast maturation.

overall_design: RNA sequencing analysis was performed in WT and Zfp281KO mouse embryos at E6.5. Genome binding/occupancy profiling of Zfp281 was performed in mouse epiblast stem cells (EpiSCs) by ChIP sequencing.

first: Xin

last: Huang


title: Transcriptional regulation of Chlamydia pneumoniae under hypoxia

city: VALENCIA

first: LOMAS LOPEZ

last: RODRIGO

address: C/ Eduardo Primo Yúfera, 3 (junto Oceanogràfic)

institute: CIPF

summary:
Purpose:
To further define the enhanced metabolic activity of Chlamydia pneumoniae under hypoxia, a transcriptome screen was performed. Next-genration sequencing (NGS) has revolutioned systems-based analysis of transcriptomic pathways. The goals of this study are to compare the transcriptomic profile of C. pneumoniae, grown within HEp-2 cells under normoxic and hypoxic conditions.
Methods:
Total RNA of C. pneumoniae infected HEp-2 cells cultured under normoxia (20% O2) or hypoxia (2% O2) was isolated by NucleoSpin RNA kit (Macherey Nagel). Human rRNA was depleted by RiboZero rRNA removal kit (Epicentre) in order to enrich bacterial RNA. The tagged cDNA libraries, in the size range of 250 –400 bp, were pooled and single-read sequencing (read length 50 bp) was performed on Illumina HiSeq 2000 by BGI-Hong Kong. Illumina reads were mapped to the Chlamydia genome (GeneBank, version AE001363.1) by TopHat (version TopHat v1.0.12. Gene expression was determined by the Htseq package and data was normalized using the RPKM conversion. Finally, the differential expression analysis was done using the Bioconductor package NOISeq version 2.6.0. The NOISeq-sim function included in the package allows for differential expression estimates in absence of replication by simulating replicates considering that reads counts follow a multinominal distribution.
Results:
Ranking of differentially expressed chlamydial genes by NOISeq-sim revealed 153 upregulated and 18 downregulated candidate genes. The expression profile of selected genes was validated using qRT-PCR. Most of the upregulated genes under hypoxia belong to the transcriptional and translational machinery or have unknown function. Moreover, transporters show increased expression under hypoxia such as the ATP/ADP translocase (Cpn0614) responsible for NAD uptake. Within the group of metabolic genes, numerous genes belonging to the energy metabolism and nucleotide metabolism were upregulated under hypoxia. Furthermore, thioredoxin reductase (trxB) belonging to the chlamydial redox system was upregulated.
Conclusions:
Our study represents the first detailed transcriptomic analysis of C. pneumoniae infected HEp-2 cells under hypoxic conditions, generated by RNA-seq technology. Our results show an increased expression of trxB, a part of the chlamydial redox system, which might reduce reactive oxygen species (ROS) under hypoxia. Interestingly, infection of hypoxic cells indeed resulted in a decreased ROS generation compared to hypoxic non-infected cells. We conclude that enhanced growth of C. pneumoniae is the result of hypoxia-induced mitochondrial dysfunction and the associated metabolic switch. Particularly, mitochondrial hyperpolarization is a central mechanism which promotes C. pneumoniae growth under hypoxic conditions. Further, our data imply that mitochondrial dysfunction could also play a major role for other intracellular pathogens, since mitochondrial dysfunction usually occurs under hypoxia and could thereby influence intracellular growth.

overall_design: Bacterial mRNA profiles of normoxic and hypoxic conditions were generated by deep sequencing, without replicates, using Illumina Hiseq 2000 by BGI Hong-Kong

first: Rodrigo

last: Lomas

first: Ana

last: Conesa

first: Nadja

last: Käding

first: Inga

last: Kaufhold

first: Constanze

last: Müller

first: Marta

last: Szaszak

first: Kensuke

last: Shima

first: Thomas

last: Weinmaier

first: Philippe

last: Schmitt-Kopplin

last: Rattei

first: Jan

last: Rupp


title: Real-time observation of light-controlled transcription in living cells

status: Public on Nov 22 2017

city: Heidelberg

first: Nick

last: Kepper

address: Im Neuenheimer Feld 267 - BQ24

department: Chromatin Networks

institute: German Cancer Research Center

summary: Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed BLInCR (Blue Light-Induced Chromatin Recruitment) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a reporter gene cluster in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ~2 minutes after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models for transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.

overall_design: RNA-Seq of untransfected, mock transfected and sample transfected U2OS2-6-3 cells

first: Anne

last: Rademacher

first: Sabrina

last: Schumacher

first: Karsten

last: Rippe


title: The pluripotency factor Zfp281 coordinates transcriptional and epigenetic control of epiblast maturation [ChIP-seq]

city: New York

first: Jianlong

last: Wang

institute: Columbia University Irving Medical Center

last: Huang