title: A comparison between single cell RNA sequencing and single molecule RNA FISH for rare cell analysis

city: Philadelphia

first: Eduardo

last: Torre

department: Bioengineering

institute: University of Pennsylvania

summary: We use single molecule RNA FISH measurements of 26 genes in thousands of melanoma cells to provide an independent reference dataset to assess the performance of the DropSeq and Fluidigm single cell RNA sequencing platforms. We quantified the Gini coefficient, a measure of rare-cell expression variability, and find that the correspondence between RNA FISH and single cell RNA sequencing for Gini, unlike for mean, increases markedly with per-cell library complexity up to a threshold of ~2000 genes detected. A similar complexity threshold also allows for robust assignment of multi-genic cell states such as cell cycle phase.

overall_design: Comparison of gene expression estimates obtained through 3 different platforms (RNA FISH, DropSeq, and Fluidigm's C1 HT IFC) using the same cell line.

first: Hannah

last: Dueck

first: Sydney

last: Shaffer

first: Janko

last: Gospocic

first: Rohit

last: Gupte

first: Roberto

last: Bonasio

first: Junhyong

last: Kim

first: John

last: Murray

first: Arjun

last: Raj


title: Facultative CTCF sites moderate mammary super-enhancer activity and regulate juxtaposed gene in non-mammary cells [4C-seq]

status: Public on May 25 2017

city: Bethesda

first: Michaela

last: Willi

address: 9000 Rockville Pike

department: NIDDK

country: USA

institute: NIH

summary: Precise spatiotemporal regulation of genetic programs, driven by cellspecific super-enhancers, is paramount for the function of cell lineages. Studies have suggested that insulated neighborhoods, formed by the zincfinger protein CTCF, sequester genes and their associated enhancers thus preventing them from trespassing on off-target genes. Although this could explain the enhancer-gene-specificity conundrum, there is limited genetic evidence that the search space of cell-specific super-enhancers is constrained by CTCF. We have addressed this question in the Wap locus with its exceptional mammary-specific super-enhancer, which is separated by five CTCF sites from neighboring genes. Three of these sites are positioned between the Wap super-enhancer and the widely expressed Ramp3. Enhancer deletions demonstrated that the Wap super-enhancer controls Ramp3 expression despite the presence of three parting CTCF sites. Individual and combinatorial deletions of these CTCF sites revealed cell-specific functions of the conserved anchor site. Although unable to block super-enhancer activity, it muffled its impact on Ramp3 in mammary tissue. Unexpectedly, this CTCF site was obligatory for Ramp3 expression in cerebellum, suggesting the coinciding presence of regulatory elements. While our results suggest a surprisingly limited in vivo role for a CTCF anchor in blocking a mammary-specific super-enhancer, they also implicate this site in cerebellum-specific gene activation. Our study illustrates additional complexities of CTCF sites supporting tissue-specific functions.

overall_design: WT and STAT5A-delS3

first: Hye

middle: K

last: Lee

first: Lothar

last: Hennighausen


title: Molecular phenotyping empowers drug discovery: a proof-of-concept study

status: Public on May 24 2017

city: Basel

first: Jitao David

last: Zhang

address: Grenzacherstrasse 124

department: Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel

country: Switzerland

institute: F. Hoffmann-La Roche

type: Expression profiling by high throughput sequencing

summary: Today, novel candidate therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. We present a strategy that allows biological relevance to be assessed in the early stages of drug discovery. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. The technique was applicable to compounds with a range of binding specificities and detected false-positive hits missed by classical phenotypic assays. Our results advocate application of molecular phenotyping in drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade.

overall_design: Molecular Phenotyping after 12h of compound treatment; A total number of 80 compounds were profiled with molecular phenotyping, along with four control conditions: media control, which represents the healthy status; GEC, which mimicks the stress condition in T2D patients; BM, a cocktail of bosentan, an enthothelin receptor antagonist, and mifepristone, a progesterone receptor antagonist,  which is supposed to reverse the disease phenotype; and GEC+BM, which mimicks a successful reversal of the phenotype from the stress condtion. The compounds were screened on six plates (A-F), with one replicate for each compound and one replicate for each control on each plate. Some compounds that failed the first screening run were tested in two cherry-picking replication plates (R1 and R2). Calcium channel inhibitor hits were then tested in phenotypic high-content microscopy assays with two or three biological replicates each.

first: Erich

last: Kueng

first: Ulrich

last: Certa

first: Martin

last: Ebeling

first: Jitao

middle: D


title: Transcriptomic Dynamics during Differentiation Process of Human Pluripotent Cells into Hepatocyte-like Cells

city: los angeles

first: Qin

last: An

state: CALIFORNIA

address: Room 6554 Gonda Building, UCLA

department: Department of Human Genetics

institute: ucla

summary: Examine characteristics of hESCs and hiPSCs during directed in-vitro differentiation into HLCs.

overall_design: We generated HLCs from both hESCs and hiPSCs by directed in-vitro differentiation, and analyzing their transcriptome at representative time points during the differentiation using RNA sequencing.

first: Yun

last: Feng

first: Rongfeng

last: Jiang

first: Xiaoqian

first: Yunwen

last: Zheng

first: Xianmin

last: Zhu

first: Guoping

last: Fan

first: Zhigang

last: Xue


title: RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome

status: Public on May 23 2017

city: Boulder

first: Roy

middle: Robert

last: Parker

state: CO

address: 3415 Colorado Ave

department: Chemistry and Biochemistry

institute: University of Colorado at Boulder

summary: Ribonucleoprotein (RNP) granules are non-membrane bound organelles thought to assemble by protein-protein interactions of RNA-binding proteins.  We present evidence that a wide variety of RNAs self-assemble in vitro into either liquid-liquid phase separations or more stable assembles, referred to as RNA tangles. Self-assembly in vitro is affected by RNA length, ionic conditions, and sequence.  Remarkably, self-assembly of yeast RNA in vitro under physiological salt mimics the composition of mRNAs within yeast stress granules. This suggests that the biophysical principles that drive RNA self-assembly in vitro contribute to determining the stress granule transcriptome. Consistent with RNA self-assembly contributing to RNP granule formation, an excess of purified RNA injected into C. elegans syncytium coalesces into droplet-like assemblies.  We suggest that diverse RNA-RNA interactions in trans contribute to RNP granule formation and may explain the prevalence of large RNA-protein assemblies in eukaryotic cells.

overall_design: RNA mixtures were assembled at 150mM NaCl, 10%PEG, 1mM MgCl2 and 150ug/mL total yeast RNA for comparison against total yeast RNA. RNA assemblies were pelleted and sent to the University of Colorado BioFrontiers Institute Next-Gen Sequencing Core Facility for Ribo-Zero treatment, library construction and NextSeq run.

first: Briana

last: Van Treeck

first: David

middle: S

last: Protter

first: Tyler

last: Matheny

first: Christopher

last: Link


title: Enforcement of developmental lineage specificity by transcription factor Oct1 (ChIP-Seq)

city: Salt Lake City

first: Brett

last: Milash

state: UT

department: Center for High Performance Computing

institute: University of Utah

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Embryonic stem cells (ESCs) co-express Oct4/POU5F1 and an Oct4 paralog known Oct1/POU2F1. To study the role of Oct1 in embryonic stem cell transcriptional control and pluripotency, we constructed germline and inducible-conditional Oct1 deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal, growth rates and metabolic signatures. However loss of Oct1 results in defective lineage specification. ESCs lacking Oct1 fail to form beating cardiomyocytes in culture, generate neurons poorly, form smaller, less differentiated teratomas, and are incapable of generating chimeric mice. Upon RA-mediated neuronal differentiation, Oct1 deficient cells fail to properly induce developmentally poised genes. Simultaneously, genes for alternative developmental pathways, most notably for the development of extra-embryonic tissues, are inappropriately expressed. ChIP experiments show that Oct1 does not occupy pluripotency genes or differentially expressed developmental targets in ESCs. Additionally, Oct1 occupies developmental targets as cells differentiate and Oct4 is lost. These results are consistent with a role for Oct1 in promoting the expression of lineage-specific genes in differentiating cells while simultaneously repressing genes specific for alternative lineages.

overall_design: Examination of occupancy of Oct1 and Oct4 in mouse embryonic stem cells

first: Zuolian

last: Shen

first: Arvind

last: Shakya

first: Jinsuk

last: Kang

first: Marcin

last: Tabaka

first: Elke

last: Jarboe

first: Aviv

last: Regev

first: Dean

last: Tantin


title: Enforcement of developmental lineage specificity by transcription factor Oct1

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

overall_design: Refer to individual Series


title: Selective expression of long non-coding RNAs in a breast cancer cell progression model

city: Burlington

first: Jonathan

middle: AR

last: Gordon

state: VT

address: 89 Beaumont Ave Given E209

department: Biochemistry

institute: University of Vermont

type: Non-coding RNA profiling by high throughput sequencing

summary: Long non-coding RNAs are emerging as key regulators of cancer cell activities. In this study, our goal was to perform a stringent profiling of breast cancer cell lines that represented a progression series. We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (early-stage), and MCF-10CA1a (malignant). We have identified expressed mRNAs and lncRNAs within each cell type. From our analysis, we identified a lncRNA of interest, IGFL2-AS1, which was knocked down in the MCF-10AT1 for validation of function.

overall_design: We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (early-stage), and MCF-10CA1a (malignant).  From our analysis, we identified a lncRNA of interest, IGFL2-AS1, which was knocked down in the MCF-10AT1 for validation of function.

first: Tracy

last: Kirsten

first: Tye

last: Coralee

first: Boyd

last: Joseph

first: Lian

last: Jane

first: Stein

last: Gary


title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

city: Muenster

first: Benedikt

middle: Sebastian

last: Nilges

address: Von-Esmarch-Straße 54

department: RNA Biology Group

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: Many cellular proteins are post-translationally modified by methylation of lysine residues. This has been most intensively studied in the case of histone proteins, where lysine methylations in the flexible tails are determinants of chromatin state and gene expression. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. While the majority of identified human KMTs belong to the SET (Su(var)3-9, Enhancer-of-zeste, Trithorax)-domain class of methyltransferases (MTases), several recently discovered KMTs belong to a different MTase class, the seven-beta-strand (7BS) MTases. Here, we have investigated an uncharacterized human 7BS MTase currently annotated as being part of the endothelin converting enzyme 2 (ECE2). However, transcriptomics data indicate that this MTase is not part of ECE2, and should be considered a separate protein. Combining in vitro enzymology and analyses of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we demonstrate that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site, and we show the importance of this motif for catalytic activity.

overall_design:
Ribosome profiling and RNAseq data for wt and  EEF1AKMT4 knockout cells.

This study includes the re-use of samples GSM2445294 and GSM2445295 from GSE93133. These two samples have been re-accessioned here so that the complete study is available from the SRP ID.

first: Magnus

middle: E

last: Jakobson

first: Jędrzej

middle: M

last: Małecki

first: Anders

last: Moen

first: Sebastian

middle: A

last: Leidel

first: Pål

last: Falnes


title: Chromatin structure and CTCF across the MCF10 breast cancer progression series (HiC)

type:
Other
Third-party reanalysis

summary: Transcription and processing of histone mRNAs occurs at subnuclear domains known as Histone Locus Bodies (HLBs). Although it is known that higher-order chromatin organization is dysregulated in cancer, structural alterations within HLB across breast cancer progression are unclear. Differential expression across known as MCF10, a basal triple negative breast cancer progression model, followed by pathway analysis demonstrated many alterations in signaling cascades known to be related to proliferation. Positional gene enrichment identified the hist1 gene locus at chromosome 6p22 as the most significantly altered cluster of genes from the normal-like MCF10A to premalignant MCF10AT1. The malignant MCF10CA1a had a more varied expression pattern across the hist1 locus. Within this region there are three subclusters of hist1 genes which were generally upregulated while intervening genes were more frequently downregulated. Extending these findings to TCGA breast tumors revealed that normal adjacent tissue had lower expression within the subclusters than their matched tumor samples. Moreover, the expression pattern within the hist1 locus comparing normal versus tumors recapitulated a very similar pattern to that comparing 10A to AT1 or CA1a. Since the hist1 locus is transcribed and processed at a specific subnuclear microenvironment, the higher order chromatin organization of this locus may be a critical component of its regulation. We addressed whether CTCF, a key chromatin organizing protein, may play a role in organizing the hist1 HLB. Immunofluorescence for CTCF along with NPAT, a protein which decorates this locus, identified that CTCF may tether the hist1 HLB to the nuclear matrix. All three subclusters reside within a single topologically associating domain wherein interacting loops are bound by CTCF. Inter-subcluster interactions are lost in the premalignant AT1. Consistent with the varied expression across the hist1 HLB in CA1a, inter-subcluster interactions are regained in the malignant state. These data suggest a transient state in highly proliferative breast cancer cells in which the hist1 HLB is dynamically reorganized.

overall_design:
HiC

------------------------

title: MCF10A Hi-C GSM1631184
source name: mammary gland/breast epithelial cell
characteristics: cell type: basal epithelial, non-tumorigenic
characteristics: neoplasia type: fibrocystic disease
characteristics: ATCC ID: ATCC CRL-10317
description: reanalysis of GSM1631184/SRR1909069
processed data file: MCF10A_pooled_40000_iced.matrix.gz
processed data file: HiCPro_40000_matrix_coordinates.bed
processed data file: MCF10A_pooled.allValidPairs.hic

first: Andrew

last: Fritz

first: Joseph

last: Boyd

first: Coralee

last: Tye

first: Jane

last: Lian

first: Gary

last: Stein


title: Enforcement of developmental lineage specificity by transcription factor Oct1 (RNA-Seq)

first: Brett

last: Milash

overall_design: Two genotypes (WT and an Oct1 inducible knockout) in mouse embryonic stem cells were compared at two different stages of differentiation (day 0 and day 14). Experiment performed in triplicate, with 12 samples total.

first: Zuolian

last: Shen

first: Arvind

last: Shakya

first: Jinsuk

last: Kang

first: Marcin

last: Tabaka

first: Elke

last: Jarboe

first: Aviv

last: Regev

first: Dean

last: Tantin


title: Mediator binds to boundaries of chromosomally interacting domains and to proteins involved in DNA looping

first: Razvan

middle: V.

last: Chereji

state: Maryland

address: 6 Center Drive, Room 2A14

department: NICHD

institute: National Institutes of Health

summary: We have combined biochemical purification of Mediator from chromatin with ChIP-sequencing to reveal Mediator occupupancy to DNA globally and to identify proteins interacting specifically with Mediator in chromatin. We find that Mediator occupy strong chromosomally interacting domain (CID) boundaries and nearly all tRNA genes. Purification of Mediator from chromatin shows that it interacts with proteins and protein complexes that have been shown to interact with CID boundaries such as RSC, Ssu72 and histone H4. We also show specific interaction between Mediator and the Arp2/Arp3, CPF, CF 1A and LSm, complexes in chromatin. These factors are involved in mRNA 3'-end processing, gene looping, actin assembly and mRNA decay.

overall_design: We assesed binding of Mediator to chromatin by ChIP-Seq. We sequenced ChIP DNA libraries prepared from Myc- and TAP-tagged Med3, Med7, Med14, Med15, Med17, Med19 and CycC strains.

middle: V

first: Vasudha

last: Bharatula

first: Nils

last: Elfving

first: Jeanette

last: Blomberg

first: Miriam

last: Larsson

first: Alexandre

last: Morozov

first: James

middle: R

last: Broach

first: Stefan

last: Björklund


title: NFAT1 binding genome-wide in memory-like CTLs

city: New York

first: Tarmo

last: Äijö

address: 162 5th Avenue

department: Center for Computational Biology

institute: Flatiron Institute

summary: In this study we investigated the role of NFATs in the regulation regulation of gene expression and transcriptional elongation in vitro.

overall_design: Determination of NFAT1 binding sites in CD8 T cells in vitro

first: Gustavo

middle: J

last: Martinez

first: Matthew

last: Pipkin

last: Aijo

first: Anjana

last: Rao


title: Hi-C As A New Tool For Precise Detection And Characterization Of Chromosomal Rearrangements And Copy Number Variation In Human Tumors

city: Cambridge

first: Steven

middle: William

last: Wingett

state: Cambs

address: Francis Crick Avenue, Cambridge Biomedical Campus

department: Cell Biology

country: United Kingdom

institute: MRC Laboratory of Molecular Biology

summary: Chromosomal rearrangements are prevalent events in the human population, occurring both constitutionally in the general population and somatically in the majority of cancers. Detection of balanced rearrangements, such as reciprocal translocations and inversions, is troublesome with many rearrangements remaining undetected. This is particularly detrimental in oncology where rearrangements play diagnostic and prognostic roles and can influence choice and efficacy of treatment protocols. New methods to detect and characterize balanced rearrangements are therefore required for both research and clinical applications. Here we describe the use of the chromosome conformation capture derived method Hi-C as a tool for detection of both balanced and unbalanced chromosomal rearrangements in cell lines and human tumor samples, with the ability to detect gene fusions and define chromosomal breakpoints to base pair resolution. In addition, we show that copy number information can also be obtained from the same data, allowing rearrangements, gains, amplifications and deletions of genomic regions to be detected in a single experiment.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Performed Hi-C on two human lymphoblastoid cell lines with known chromosomal translocations (FY1199 and DD1618) and on a transformed mouse cell line (EKLF). In addition, performed Hi-C on six human brain tumours: five glioblastomas ( GB176, GB180, GB182, GB183 and GB238) and one anaplastic astrocytoma (AA86). Hi-C was also performed in a human cell line control (GM07017).  QDNAseq was performed on the AA86, GB176, GB180, GB182, GB183 and GB238 sampes.

first: Louise

last: Harewood

middle: W

first: Danita

last: Pearson

last: Eldridge

last: Schoenfelder

first: V

middle: P

last: Collins

first: Peter

last: Fraser


title: Next Generation Sequencing Comparison of Wild Type and Whsc1-/- Activated B-cell Transcriptomes

city: Madrid

first: Cesar

last: Cobaleda

address: c/Nicolas Cabrera, 1

country: Spain

institute: CBM, CSIC

summary: Whsc1 gene codes for a SET domain-containing H3K36 dimethylase, whose activity has been suggested, in ex vivo cell culture experiments, to control many aspects of DNA and RNA processing (replication, repair, transcription, etc). Its precise function in vivo is still unclear. Here, we use RNA-seq transcriptome analysis to study the changes in gene expression in the absence of Whsc1. Our results show that, in the experimental system used, loss of Whsc1 caused massive changes in genes affecting many fundamental cellular processes, from cell cycle to ribosome synthesis, DNA repair, replication, etc.

overall_design: Whsc1-KO mice are embryonic lethal. We therefore took hematopoietic cells from fetal liver of WT and Whsc1-KO embryo littermates and injected them in to lethally irradiated RAG1-KO recipients and allowed the generation of a full Whsc1-KO hematopoietic system. Then, WT and Whsc1-KO B cells were obtained from the spleen and stimulated with LPS to induce proliferation and class switch recombination. Flow cytometry and cell cycle analyses (among others) showed the existence of serious proliferative alterations in Whsc1-KO cells. Then, we performed paired-end RNAseq analyses of 7 independent WT and 6 independent Whsc1-KO biological replicates and we used these data to identify differentially expressed genes and pathways regulated by Whsc1 in B cells.


title: Characterisation of mRNA expression and replication fork stalling in copper-treated yeast

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

summary: Here we analyse steady state mRNA levels and yH2A-marked replication fork stalling sites in S. cerevisiae

overall_design: Histone modification (yH2A, gammaH2A) and transcriptome (mRNA-Seq) from wildtype BY4741 cells grown in YPD and treated for 4 hours with 0 or 1mM CuSO4

first: Ryan

last: Hull

first: Cristina

last: Cruz

first: Jonathan

last: Houseley


title: Targeting Taxane-Platin Resistant Lung Cancers with JumonjiC Lysine Demethylase Inhibitors

city: Dallas

first: Ralf

last: Kittler

state: Texas

address: 5323 Harry Hines Blvd

institute: University of Texas Southwestern Medical Center


title: Gene expression changes upon NUSAP1 over- or under-expression in PC-3 or DU145 cells.

city: Stanford

first: James

middle: D.

last: Brooks

state: CA

address: 300 Pasteur Drive

department: Urology

institute: Stanford University

summary: By transcriptome (RNA-Seq) analysis of PC-3 or DU145 prostate cancer cell lines over- or under-expressing NUSAP1, we determined genes that become differentially expressed upon expression changes of NUSAP1. Ingenuity Pathway Analysis revealed that the differentially expressed genes correlated with increased tumor progression and are involved in functions that include cancer, cellular movement, and cell morphology

overall_design: Lentiviral infections were used to over- or under-express NUSAP1 or controls in triplicate in PC-3 or DU145 cell lines. Seventy-two or ninety-six hours after infection, total RNA was extracted and purified. The sequencing libraries were prepared with the TruSeq RNA Sample Preparation Kit v2 (Illumina) or TruSeq Stranded mRNA Sample Preparation Kit (Illumina) as directed by the manufacturer’s protocol.  Pooled libraries were run on a Hiseq 2000 Sequencing System (Illumina) with 101 base pair single-end reads.

first: Catherine

last: Gordon


title: Targeting Taxane-Platin Resistant Lung Cancers with JumonjiC Lysine Demethylase Inhibitors (ChIP-Seq)

summary: Characterization of histone 3 lysine 4 and lysine 27 trimethylation changes upon development of taxane-platin drug resistance in NSCLC cells and evaluation of these histone modifications after treatment with Jumonji KDM inhibitors, JIB-04 and GSK-J4.

overall_design: Comparison of H3K4me3 and H3K27me3 profiles between H1299 Parental cells (chemo-sensitive) and H1299 T18 cells (taxane-platin resistant), and assessment of Jumonji KDM inhibitors on H1299 T18 cells (JIB-04 treated vs DMSO treated, and GSK-J4 treated vs. DMSO treated).

first: Maithili

last: Dalvi

first: Paul

last: Yenerall

first: Rahul

middle: K

last: Kollipara

first: Elisabeth

last: Martinez

first: John

last: Minna


title: Targeting Taxane-Platin Resistant Lung Cancers with JumonjiC Lysine Demethylase Inhibitors (RNA-Seq)

summary: Characterization of gene expression changes upon development of taxane-platin drug resistance in NSCLC cells and further, upon treatment of these resistant cells with the Jumonji KDM inhibitor, GSK-J4.

overall_design: Comparison of gene expression changes between H1299 Parental cells (chemo-sensitive) and H1299 T18 cells (taxane-platin resistant), and comparison of H1299 T18: GSK-J4 treated vs. H1299 T18: DMSO control.

middle: D


title: Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance

status: Public on May 22 2017

city: Smithville

first: Kei-ichi

last: Takata

state: TX

address: 1808 Park Road 1C

department: Department of Epigenetics and Molecular Carcinogenesis

institute: The University of Texas MD Anderson Cancer Center

summary: Whole genome transcriptome analysis by RNAseq was performed to determine whether Poln disruption influences gene expression in testis from PolnΔEx2/ΔEx2 mice.

overall_design: Testis mRNA profiles of 68-day old wild type (WT) and Poln∆Ex2/∆Ex2 (Poln) mice were generated by Illumina HiSeq 2000.

first: Shelley

last: Reh

first: Matthew

last: Yousefzadeh

first: Yoko

first: Yue

last: Lu

first: Kevin

last: Lin

first: Jianjun

last: Shen

first: Richard

last: Wood


title: Cellular Interplay and Cytokine Hierarchy Cause Pathological Cardiac Hypertrophy in RAF1-Mutant Noonan Syndrome

city: Toronto

first: Pamela

last: Ohashi

state: Ontario

address: 610 University Ave

country: Canada

institute: Princess Margaret Cancer Centre

summary: Noonan syndrome (NS) is caused by mutations in RAS/ERK pathway genes, and is characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1 L613V/+ knock-in mice. Here, using inducible Raf1 L613V expression, we show that LVH results from the interplay of cardiac cell types. Cardiomyocyte Raf1 L613V enhances Ca 2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1 L613V expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1 L613V causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine hierarchy (TNFα->IL6) from Raf1 L613V -expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, post-natal TNFα inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. We conclude that NS cardiomyopathy involves cardiomyocytes, ECs, and fibroblasts, TNFα/IL6 signaling components represent potential therapeutic targets, and abnormal EC signaling might contribute to other forms of LVH.

overall_design: control  vs. Raf1 L613V/+ knock-in cardiomyocytes

first: Azin

last: Sayad

first: Connie

last: Yin

first: Benjamin

last: Neel


title: Identification of preoptic sleep-promoting neurons based on projection target

city: Seattle

first: Thuc Nghi

last: Nguyen

state: WA

address: 615 Westlake Ave N

institute: Allen Institute for Brain Science

summary: The preoptic area (POA) of the hypothalamus is known to be crucial for sleep generation, but the spatial intermingling of sleep- and wake-promoting neurons makes it difficult to dissect the sleep control circuit. Here we identified a population of POA sleep-promoting neurons based on their projection target. Using a lentivirus for retrograde labeling with channelrhodopsin-2 (ChR2) followed by optogenetic manipulation and recording, we found that the POA GABAergic neurons projecting to the tuberomammillary nucleus (TMN) are both sleep active and sleep promoting. Cell type- and projection-specific rabies tracing revealed the presynaptic inputs to these neurons, including an amygdala GABAergic input that promotes wakefulness. Using single-cell RNA-seq, we identified several molecular markers for these neurons, and optogenetic activation of the POA neurons labeled by these markers confirmed their sleep-promoting effects. Together, these findings define a group of sleep-promoting neurons functionally, anatomically, and genetically.

overall_design: Single-cell RNA-Seq of retrogradely-labeled POA neurons projecting to the tuberomammillary nucleus (TMN).

first: Bosiljka

last: Tasic

first: Shinjae

last: Chung

first: Yang

last: Dan


title: Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity

city: Cleveland

first: Ann

last: Harris

state: OH

address: 10900 Euclid Ave

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), but are not good predictors of lung disease phenotype. Genome-wide association studies (GWAS) previously identified additional regions of the genome associated with CF disease severity. One of these, at chromosome 11p13, is an intergenic region between Ets homologous factor (EHF) and Apaf-1 interacting protein (APIP). Our goal was to determine the functional significance of this region, which is predicted to be regulatory, since it lacks annotated genes. To identify cis-regulatory elements within the chr11p13 region in lung epithelial cells, we first mapped open chromatin, using DNase I digestion and deep sequencing (DNase-seq). Next, histone modifications associated with active chromatin, H3K4me1 and H3K27ac, were revealed by chromatin immunoprecipitation. Predicted enhancer elements were assayed in bronchial epithelial cells using luciferase reporter genes. Two elements showed strong enhancer activity for the promoters of both EHF and the 5’ adjacent gene E47 like ETS transcription factor 5 (ELF5). No enhancers of the APIP promoter were found. Next, to determine direct physical interactions between cis-regulatory elements and gene promoters within 11p13 we used circular chromosome conformation capture (4C-seq). 4C-seq confirmed the enhancer-promoter associations, identified additional interacting elements and defined the boundaries of the topologically associated domains (TADs) at 11p13, which are enriched for CTCF. No strong interactions were observed with the APIP promoter, which lies outside of the main TAD encompassing the GWAS signal. These results focus attention on the role of EHF in modifying CF lung disease severity.

overall_design: To identify regulatory elements at the 11p13 cystic fibrosis modifier region, DNase-seq was performed on 16HBE14o- transformed lung bronchial epithelial cells, and ChIP-seq for H3K4me3, H3K27me3, H3K9me2, H3K9me3, and H3K36me3 was performed in Calu3 lung adenocarcinoma cells and primary human bronchial epithelial cells

first: Lindsay

last: Stolzenburg

first: Rui

last: Yang

first: Jenny

middle: L

last: Kerschner

first: Sara

last: Fossum

last: Xu

first: Andrew

last: Hoffmann

first: Sujana

last: Ghosh

first: Kay-Marie

last: Lamar

first: Sarah

last: Wachtel

first: Shih-Hsing

last: Leir


title: Transcriptome profiling of Peyer’s patch naive and germinal center B cells

first: Ángel

middle: F

last: Álvarez-Prado

address: Melchor Fernández Almagro, 3

institute: Centro Nacional de Investigaciones Cardiovasculares

summary:
Germinal center (CD19+Fas+GL7+) and naive (CD19+Fas-GL7-) B cells were sorted from Peyer's patches of littermate 12 weeks old WT C57BL/6 mice.
Three biological replicates were analyzed, each composed of a pool of 5 female mice.
RNA was purified from pellets of 2-2.5x10^⁴ cells and sequencing libraries were prepared from 100ng of total RNA per replicate.

overall_design: Transcriptional profiling of germinal center and naive B cells from Peyer’s patches of WT mice.

first: Almudena

last: Ramiro


title: CTCF depletion effect in B cells

first: Manuel

last: Gomez

state: Madrid

institute: CNIC

summary: Role of CTCF in activated B cells.

overall_design: Transcriptome profiling of  CTCF deficient and proficient activated in vitro B cells.

first: Arantxa

last: Pérez-García

first: Ester

last: Marina-Zárate

first: Jose

last: Ligos

first: Niels

last: Galjart


title: morc-1 regulates transgenerational heterochromatin maintenance at nuclear RNAi targets

city: Baltimore

first: Mallory

middle: Ann

last: Freeberg

state: MD

address: 3400 N Charles St

department: Department of Biology

institute: Johns Hopkins University

summary: The germline is an immortal cell lineage and therefore must accurately transmit genetic and epigenetic information over infinite cell divisions to ensure fertility and species survival. Endogenous siRNAs (endo-siRNAs), which are highly expressed in the parental germline and inherited in their progeny, represent one mechanism by which epigenetic information can be transmitted from one generation to the next. In C. elegans, this is accomplished by the nuclear RNAi pathway, which ensures robust inheritance of endo-siRNAs and of target gene silencing through deposition of H3K9me3 marks at target loci. How these marks are maintained in subsequent generations is unknown. Here, we show that the C. elegans micro-orchidia homolog, MORC-1, functions downstream of endo-siRNAs to mediate target silencing and maintenance of siRNA-dependent H3K9me3 marks. Like the established members of the nuclear RNAi pathway, morc-1 is essential for germline maintenance and thus morc-1(-) mutants exhibit a mortal germline phenotype. Furthermore, morc-1(-) mutants exhibit severe decondensation of germline chromatin. Through a forward genetic screen, we have identified mutations in the gene encoding MET-1, a H3K36 histone methyltransferase, that potently suppress morc-1(-) germline mortality. We show that morc-1(-) mutants exhibit progressive, met-1-dependent enrichment of H3K36me3 and that mutation of met-1 can restore germline chromatin organization in late generation morc-1(-) mutants. Our data suggest that MORC-1 is required to repress MET-1 activity at nuclear RNAi target genes and that in the absence of this repression, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality.

overall_design: We performed replicate small RNA-seq, mRNA-seq, and ChIP-seq of two histone methylation marks (H3K9me3, H3K36me3) in morc-1 mutant animals with wild-type and hrde-1 mutant animals as controls. In addition, ChIP-seq was performed in the same strains in a met-1 mutant background.

first: Natasha

last: Weiser

first: Sam

middle: G

last: Gu

first: Natallia

last: Kalinava

first: Taiowa

last: Montgomery

first: Kristen

middle: C

last: Brown

first: Suhua

last: Feng

first: Steven

last: Jacobson

first: John

last: Kim


title: Identification of Pax7 target genes in muscle progenitor cells from young mice

city: Shenzhen

first: Kun

last: Sun

address: Rm B505, No. 9 Duxue Road, Nanshan District

department: Cancer Research

country: China

institute: Shenzhen Bay Laboratory

summary: To identify Pax7 target genes in muscle progenitor cells, we compared the transcriptome profiles of muscle progenitor cells from young mice (P12) with and without Pax7.

overall_design: Isolate muscle progenitor cells from young mice (control and Pax7-KO), extract total RNA and subject them to RNA-seq

first: Yarui

last: Diao

first: Zhenguo

last: Wu

first: Huating

last: Wang


title: Effects of XPC overexpression on global methylation

city: Berkeley

first: Robert

last: Tjian

address: University of California, Berkeley 450 Li Ka Shing #3370

department: MCB

institute: UC Berkeley

type: Methylation profiling by high throughput sequencing

summary: The study examines the impact of XPC overexpression on DNA demethylation in human dermal fibroblasts (HDFs) and during the early phase of somatic cell reprogramming (pre-iPSCs). We profiled the 5mC status in wild type and XPC gain-of-function HDFs and pre-iPSCs on a genome-wide scale by MeDIP-seq. XPC overexpression results in a loss of DNA methylation in both HDFs and pre-iPSCs. In XPC gain-of-function cells, not only does DNA methylation occur in fewer regions compared to control WT cells, but overall methylation levels are lower than in WT cells.

overall_design: MeDIP-seq of wild type and XPC-overexpressing HDFs and pre-iPSCs (7 days into reprogramming). We prepared libraries from each MeDIP input as a control. No sample DNA was recovered for IgG IPs, indicating very little background binding was occurring.

first: Jaclyn

last: Ho

first: Claudia

last: Cattoglio


title: Hhex regulates HSC self-renewal and stress hematopoiesis via repression of Cdkn2a

city: Clayton

first: Wei

last: Shi

state: Victoria

address: Wellington Rd

country: Australia

institute: Monash University

summary: The Hematopoietically-expressed homeobox transcription factor (Hhex) is important for the maturation of definitive hematopoietic progenitors and B-cells during development. We have recently shown that in adult hematopoiesis, Hhex is dispensable for maintenance of hematopoietic stem cells (HSCs) and myeloid lineages but essential for the commitment of Common Lymphoid Progenitors (CLPs) to lymphoid lineages. However, whether Hhex plays a role in HSC self-renewal and myeloid expansion during hematopoietic stress is unknown. Here we show that during serial bone marrow transplantation, Hhex-deleted HSCs are progressively lost, revealing an intrinsic defect in HSC self-renewal. Moreover, Hhex-deleted mice show markedly impaired hematopoietic recovery following myeloablation. In vitro, Hhex-null blast colonies were incapable of replating, implying a specific requirement for Hhex in immature hematopoietic progenitors. Transcriptome analysis of Hhex-null Lin-Sca+Kit+ (LSK) cells showed that Hhex deletion leads to the deregulation of Polycomb Repressive Complex 2 (PRC2) target genes, including an upregulation of Cdkn2a locus, encoding the cell cycle repressors p16Ink4a and p19Arf. Indeed, loss of Cdkn2a restored Hhex-null blast colony replating in vitro, as well as hematopoietic reconstitution following myeloablation in vivo. Thus, HSCs require Hhex to repress Cdkn2a to enable continued self-renewal and response to hematopoietic stress.

overall_design: Transcriptional profiling of Hhex-deleted and wild-type LSK cells using RNA sequencing

first: Jacob

middle: T

last: Jackson

first: Benjamin

last: Shields

first: Matthew

last: McCormack


title: Ets homologous factor has critical roles in epithelial dysfunction in airway disease [RNA-seq]

city: Chicago

first: Ann

last: Harris

state: IL

address: 2430 N Halsted Street  Box 211

department: Human Molecular Genetics

institute: Lurie Children's Hospital of Chicago Research Center

summary: Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor. Using EHF ChIP-seq and RNA-seq after EHF depletion, we show its targets in HBE cells are enriched for genes involved in degradation of misfolded proteins, inflammation, and wound repair.

overall_design: mRNA profiles from primary human bronchial epithelial cells treated with negative control (NC) or EHF siRNA, in quintuplicate.

first: Sara

last: Fossum

first: Mutolo

last: Michael

first: Antonio

last: Tugores

first: Sujana

last: Gosh

first: Scott

last: Randell

first: Lisa

last: Jones

first: Shih-Hsing

last: Leir


title: Olfactory sensory neuron-specific IGF1R knockout in mice results in increased smell perception, insulin resistance and adiposity

city: Köln

first: Eva

last: Tsaousidou

address: Gleueler Str. 50

department: Neuronal Control of Metabolism

institute: Max-Planck-Institute for Metabolism Research

summary: Olfaction is fundamental for survival but there is little known about the connection between smell perception and metabolism. In this study we implemented IGF1R knockout mice in the olfactory sensory neurons, by olfactory marker protetin (OMP) Cre specific recombination, and investigated metabolic parameters, smell perception and transcriptome sequencing. We could demonstrate that IGF1R knockout in the olfactory sensory neurons results in enhanced smell perception, insulin resistance under normal chow diet conditions and increased adiposity in mice fed control diet. Transcriptome analysis of the olfactory epithelium revealed differential expression of markers for mature and immature olfactory sensory neurons, being down-regulated and up- regulated respectively, pointing to differentiation-dependent changes that result in increased olfactory perception. Collectively, this study provides evidence that enhanced smell perception can result in insulin resistance and increased adiposity.

overall_design: mRNA profiles of olfactory sensory neurons (OSN) extracted from homozygous tissue-specific IGF1R knockout (OMPIGF1R) and respective cotnrol mice (OMPflfl) were generated by deep sequencing, in four replicates using Illumina sequencing

first: Celine

middle: E

last: Riera

first: Jonathan

last: Halloran

first: Patricia

last: Follet

first: Oliver

last: Hahn

first: Mafalda

middle: M

last: Pereira

first: Linda

last: Engström

first: Jens

last: Alber

first: Kevin

last: Tharp

first: Courtney

last: Anderson

first: Hella

last: Brönneke

first: Brigitte

last: Hampel

first: Carlos

last: de Magalhaes Filho

first: Andreas

last: Stahl

last: Brüning

first: Andrew

last: Dillin


title: Next Generation Sequencing Comparison of Wild Type and Whsc1-/- BM sorted proB Cell Transcriptomes

first: Cesar

last: Cobaleda

summary: Whsc1 gene codes for a SET domain-containing H3K36 dimethylase, whose activity has been suggested, in ex vivo cell culture experiments, to control many aspects of DNA and RNA processing (replication, repair, transcription, etc). Its precise function in vivo is still unclear. Here, we use RNA-seq transcriptome analysis to study the changes in gene expression in the absence of Whsc1 in sorted Bone Marrow proB cells. Our results show that, in the experimental system used, loss of Whsc1 caused massive changes in genes affecting many fundamental cellular processes and also severly interefering with B cell fate specification and commitment

overall_design: Whsc1-KO mice are embryonic lethal. We therefore took hematopoietic cells from fetal liver of WT and Whsc1-KO embryo littermates and injected them in to lethally irradiated RAG1-KO recipients and allowed the generation of a full Whsc1-KO hematopoietic system. Flow cytometry analyses showed the existence of serious differentiation alterations in Whsc1-KO B cells. Then, we sorted B220+CD19+ckit+CD25-PI- proB cells form the BM of mice injected with either WT or Whsc1-/- cells andf performed  RNAseq analyses of 2 independent WT and 2 independent Whsc1-KO biological replicates and we used these data to identify differentially expressed genes and pathways regulated by Whsc1 in pro-B cells.


title: Ets homologous factor has critical roles in epithelial dysfunction in airway disease

first: Ann

last: Harris

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

overall_design: Refer to individual Series


title: Ets homologous factor has critical roles in epithelial dysfunction in airway disease [ChIP-seq]

summary: Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that EHF binding sites in lung epithelial cells are enriched at promoters and coincide with putative enhancers, marked by specific histone modifications. Using EHF ChIP-seq and RNA-seq after EHF depletion, we show its targets in HBE cells are enriched for genes involved in degradation of misfolded proteins, inflammation, and wound repair.

overall_design: Examination of EHF binding plus three histone modifications in primary human bronchial epithelial (HBE) cells. Also examined c-Jun and JunD binding in Calu-3 cells. There are two replicates of the EHF, c-Jun and JunD ChIP-seq plus an input sample (control). There is one replicate each of the three histone modifications.

first: Sara

last: Fossum

first: Mutolo

last: Michael

first: Antonio

last: Tugores

first: Sujana

last: Gosh

first: Scott

last: Randell

first: Lisa

last: Jones

first: Shih-Hsing

last: Leir


title: Characterization of RNAi genes and their impact on sRNA biogenesis, growth and development of the rice blast fungus, Magnaporthe oryzae

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

summary: RNAi is a conserved mechanism in eukaryotic organisms and has been the subject of recent attention pertaining to many animal and plant models. But research related to fungal systems is limited, especially in plant pathogenic models. The present study was undertaken to examine the involvement of RNAi components on the biogenesis of small RNAs and their impact on growth and pathogenicity of the plant pathogenic model organism Magnaporthe oryzae. Through genetic and genomic analyses, we demonstrate that an individual Dicer, an RdRP and an Argonaute are essential for the generation of sRNAs. Collectively, they are involved in biogenesis of sRNAs matching to coding regions, repeats and intergenic regions. The loss of a particular Argonaute made the fungus less virulent on barley leaves and was also essential for sRNA production. Furthermore, one specific RdRP that was not essential to sRNA generation nonetheless reduced fungal virulence. In total, ten deletion mutants were constructed in each of the putative sRNA-generating genes, as well as a double deletion mutant in the two Dicers, and comparison of sRNA and RNA-seq profiles of each mutant yielded new insights into the involvement of sRNAs in transcriptional regulation and intergenic regions in M. oryzae. Together, these data support that RNAi machinery in M. oryzae is not only involved in generation of sRNAs, but is involved in regulating various developmental processes including fungal growth and virulence.

overall_design: Genome-wide small RNA profiling was done by Illumina TruSeq sample preparation followed by high-throughput sequencing with Illumina HiSeq 2000 platform.

first: Vidhyavathi

last: Raman

first: Stacey

middle: A

last: Simon

first: Feray

last: Demirci

first: Mayumi

last: Nakano

first: Nicole

last: Donofrio


title: Genome-wide maps of histone H4 acetylation in human K562 cells in optimal growth conditions and upon heat stress

status: Public on May 21 2017

city: Stockholm

first: Anniina

last: Vihervaara

address: Tomtebodavägen 23A

department: Gene Technology

country: Sweden

institute: KTH Royal Institute of Technology

summary: Genome-wide characterization of histone H4 acetylation in K562 cells in optimal growth conditions (NHS) and upon 30 min heat stress at 42°C (HS30).

overall_design: ChIP-seq mapping of histone H4 acetylation in non-treated and heat shocked K562 cells. Input (fragmentet total chromatin) serves as a background control. The antibody (UpState 06-866) recognizes histone H4 acetylation at lysines 5, 8, 12 and 16. Ten ChIP-replicates in each condition were prepared separately, then pooled together, purified, and sequenced as a single sample with paired-end sequencing (HiSeq PE100). Please, see also GSE43579 where HSF1 and HSF2 target loci, as well as IgG controls, were prepared from the same cell populations and in parallel ChIP-experiments, but were sequenced earlier and only from single end.

first: Lea

last: Sistonen


title: PHF20 readers link methylation of histone H3K4 and p53 with H4K16 acetylation

first: Yue

last: Lu

department: Epigenetics and Molecular Carcinogenesis

institute: MD Anderson Cancer Center

summary: PHF20 is a core component of the lysine acetyltransferase complex MOF-NSL that produces the major epigenetic mark, H4K16ac, and is necessary for transcriptional regulation and DNA repair, however the role of PHF20 in the complex remains elusive. Here, we report on functional crosstalk between epigenetic readers of PHF20. We show that the PHF20 PHD finger recognizes dimethylated lysine 4 of histone H3 (H3K4me2) and represents first example of a native PHD reader selective for this modification. Biochemical and structural analyses illuminate the molecular mechanism underlying this function and explain the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a novel PHF20-mediated link between MOF HAT, p53 and H3K4me2-dependent cellular events and suggest a model for rapid spreading of H4K16ac-encriched open chromatin.

overall_design: H1792 cells were transduced with shRNAs targeting PHF20 or control non-tareting shRNAs and selected with puromycin for 4-6 days. RNA-seq was then performed to identify differentially expressed genes among different conditions.

first: Xiaobing

last: Shi


title: The integrative omics of white-rot fungus Pycnoporus coccineus reveals co-regulated CAZymes for orchestrated lignocellulose breakdown.

city: Marseille

first: Marie-Noëlle

last: Rosso

address: 163 avenue de Luminy

department: Biodiversity and Biotechnology of Fungi - BBF

country: France

institute: INRAE - Aix Marseille University

summary: White-rot basidiomycete fungi are potent degraders of plant biomass with the ability to mineralize all lignocellulose components. Recent comparative genomics studies showed that these fungi use a wide diversity of enzymes for wood degradation. In order to improve our understanding on the enzymatic mechanisms leading to lignocellulose breakdown, we analysed the early response of the white-rot  fungus Pycnoporus coccineus CIRM-BRFM310  to various lignocellulosic substrates at two time points; Day 3 and Day 7.

overall_design: Examination of mRNA levels in fungal mycelium after 3 and 7 days growth in liquid cultures on maltose, wheat straw, pine or aspen. Each culture was done in triplicate. mRNA were sequenced using 75 bp paired end reads on Illumina HighSeq-2500. The reads were mapped on the genome Pycnoporus coccineus BRFM 310 v1.0 available on the Mycocosm portal at http://genome.jgi.doe.gov/Pycco1/Pycco1.home.html

first: Marie-Pierre

last: Franquin-Gomez

first: Shingo

last: Miyauchi


title: Gene expression profiles in naïve and activated CD4+ T cells from WT and Capicua (Cic)-deficient mice

city: Pohang

first: Yoontae

last: Lee

state: Gyeongbuk

address: 77 Cheongam-Ro, Nam-Gu

department: Life Science

country: South Korea

institute: POSTECH

summary: Purpose: Next-generation sequencing (NGS) to reveal CIC target genes in CD4 T cells. The goals of this study are to compare NGS-derived CD4 T cell transcriptome in the presence or absence of CIC.

overall_design: To identify the genes regulated by CIC in CD4+ T cells, RNA-seq was performed using naive and activated CD4+ T cells in the spleen from Cic f/f and Cic f/f; Cd4-Cre mice.

first: Sungjun

last: Park


title: Nucleotide-resolution maps of nascent RNA-synthesis in human K562 cells in optimal growth conditions and upon a 30-minute heat shock at 42°C

summary: Precision Run-On Sequencing (PRO-seq) mapping of nascent RNA synthesis at a nucleotide-resolution across the human genome. The PRO-seq data has been generated from human K562 cells that have been cultured in optimal growth conditions (NHS) or that have been exposed to an acute, 30-minute at 42°C, heat treatment (HS30). The data contains two replicates in each condition (fastq files of raw sequencing data), and bigWig files where the two replicates have been combined and the data normalized to allow direct comparison of the transcription profiles. in NHS and HS30 conditions.

overall_design: Profile of nascent transcription in non-stressed and heat-stressed human K562 cells

first: Dig

middle: B

last: Mahat

first: John

last: Lis


title: Time series RNA profiling of endotheilal cells in response to atheroprone and atheroprotective flows

city: La Jolla

first: Shu

last: Chien

address: 9500 Gilman Drive

department: Institute of Engineering In Medicine

institute: University of California, San Diego

summary: To profile shear stress-regulated endothelial transcriptomes, we performed RNA-seq with HUVECs subjected to different shear flow conditions, including atheroprotective pulsatile shear (PS, 12±4 dyn/cm2) and atheroprone oscillatory shear (OS, 0.5±4 dyn/cm2), or kept as static control (ST) for four time periods (1, 4, 12 and 24 hours)

overall_design: HUVECs cultured on collagen I-coated slides were exposed to PS, OS  or ST.  RNA were extracted with TRIzol and sequenced on Illumina HiSeq platform.

first: Tse-Shun

last: Huang

first: Kuei-Chun

last: Wang

first: Sara

last: Quon

first: Phu

last: Nguyen

first: Ting-Yu

last: Chang

first: Zhen

last: Chen

first: Yi-Shuan

last: Li

first: Shankar

last: Subramanian

last: Shyy


title: Sp9 regulates medial ganglionic eminence-derived cortical interneuron development

status: Public on May 19 2017

city: shanghai

first: Zhidong

last: Liu

address: Mingdao Building, 138 Yi Xue Yuan Road

institute: fudan university

summary: In the mammalian cortex, about 60% of GABAergic interneurons, mainly including parvalbumin-expressing (PV+) and somatostatin (SST+) interneurons are generated from the medial ganglionic eminence (MGE) in the subpallium and tangentially migrate to the cortex. Here we analyze the role of the Sp9 transcription factor in regulating the development of MGE-derived cortical interneurons. We show that SP9 is widely expressed in the MGE subventricular zone (SVZ) and in MGE-derived migrating interneurons. By analyzing Sp9 null and conditional mutant mice, we demonstrate that Sp9 promotes MGE progenitor proliferation in the SVZ and is required for the normal patterning of tangential migration and the laminar distribution of MGE-derived cortical GABAergic interneurons. Loss of Sp9 function results in a ~50% reduction of MGE-derived cortical interneurons, an ectopic aggregation of MGE-derived neurons in the embryonic ventral telencephalon, and an increased ratio of SST+/PV+ cortical interneurons. Finally, we provide evidence that Sp9 regulates MGE derived cortical interneuron development through promoting expression of the Lhx6 and Lhx8 transcription factors.

overall_design: Medial ganglionic eminence mRNA profiles of embryonic 12.5 wild type (WT) and sp9 null mutant mice were generated by deep sequencing, in triplicate, using using an illumina high-seq 2500

first: Zhuangzhi

last: Zhang

first: Zhengang

last: Yang


title: YY1 haploinsufficiency causes an intellectual disability syndrome featuring transcriptional and chromatin dysfunction [RNA-seq]

city: Milano

first: Giuseppe

last: Testa

address: Via Adamello, 16

country: Italy

institute: European Institute of Oncology & University of Milano, Italy

summary: Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth retardation, feeding problems, and various congenital malformations. Our combined clinical and molecular data define the ‘YY1 syndrome’ as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from person-derived cells, using antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding, with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.

overall_design: Individuals with mutations or deletion in YY1 were identified among patients with idiopathic intellectual disability. LCLs were established from 4 of these patients (1 deletion, 2 missense mutations, and 1 non-sense mutation undergoing non-sense-mediated decay) as well as from unrelated controls, and their transcriptome were compared.

first: Michele

last: Gabriele

first: Anneke

last: Silfhout

first: Pierre-Luc

last: Germain

first: Alessandro

last: Vitriolo

first: Raman

last: Kumar

first: Jozef

last: Gecz

first: David

last: Koolen

first: Bert

last: de Vries


title: Distinct roles for matrix metalloproteinases 2 and 9 in hematopoietic stem cell emergence, migration and niche colonization

city: Boston

first: Leonard

last: Zon

state: MA

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: We report gene expression data for FACS sorted zebrafish mpeg1:mCherry + and mpx:EGFP + cells collected from whole embryos at 72 hours post fertilization (hpf). We also report gene expression data for the remaining, transgene negative, portion of these embryos.

overall_design: ~1,000 mpeg1:mCherry+; mpx:EGFP+ transgenic embryos were homogenized, filtered, and sorted using FACS into PBS, collecting >50,000 cells for each of the three populations: mpeg1:mCherry+, mpx:EGFP+ and double negative (no double positive cells were collected as there was almost no overlap between mCherry and EGFP expression).

first: Elliott

middle: J

last: Hagedorn

first: Song


title: Elf18-induced long noncoding RNA associates with Mediator to enhance expression of innate immune response genes in Arabidopsis

city: NEW YORK

first: Nam-Hai

last: Chua

state: New York

address: 1230 York Avenue

institute: The Rockefeller University

summary: Plant immune response is a complex process involving both transcriptional and posttranscriptional regulation of gene expression. Here, we identify an Arabidopsis long-noncoding (lnc) RNA, designated elf18-induced long noncoding RNA 1 (ELENA1), as a factor enhancing resistance against Pseudomonas syringe pv. tomato DC3000. ELENA1 knock-down plants show a decreased expression of PR1 and the plants are susceptible to pathogens. By contrast, plants over-expressing ELENA1 show elevated Pathogenesis-Related gene 1 (PR1) expression after elf18 treatment and display pathogen resistance phenotype. RNA-seq analysis of ELENA1 overexpressing plants after elf18 treatment confirms increased expression of defense-related genes compared to WT. ELENA1 directly interacts with Mediator subunit 19a (MED19a) and affects enrichment of MED19a on PR1 promoter. These results show that ELENA1 is a novel lncRNA that regulates PR1 expression through MED19a. Our findings uncover an additional layer of complexity implicating lncRNA in the transcriptional regulation of plant innate immunity.

overall_design: To investigate the role of ELENA1 in regulating immune response, we performed strand-specific RNA-seq (ssRNA-seq) on WT and ELENA1 OX plants grown under normal (0 h) and elf18-treated conditions (1 h, 6 h and 12 h). For each condition we used three biological replicates.

first: Jun

middle: S

last: Seo

first: Hai-Xi

last: Sun

first: Bongsoo

last: Park

first: Chung-Hao

last: Huang

first: Shyi-Dong

last: Yeh

first: Choonkyun

last: Jung


title: Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs [mRNA bisulfite sequencing]

city: Mannheim

first: Francesca

last: Tuorto

address: Ludolf-Krehl-Str. 13-17

department: Division of Biochemistry  Mannheim Institute for Innate Immunoscience (MI3)

institute: Deutsches Krebsforschungszentrum

summary: (Cytosine-5) RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We have previously established RNA bisulfite sequencing as a method for the analysis of RNA (cytosine-5) methylation patterns at single-base resolution. Furthermore, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. We have now established a computational approach that integrates tailored filtering and data-driven statistical modelling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing. Using RNAs from mouse embryonic stem cells we have performed a comprehensive methylation analysis of mouse tRNAs, rRNAs and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA. Furthermore, the catalytic activities of the Dnmt2 tRNA methyltransferase could be resolved at single-base resolution. Of note, mRNAs were found to be very sparsely methylated or not methylated at all, which provides an important reference for further studies. Our approach can be used to profile (cytosine-5) RNA methylation patterns in many experimental contexts be important for understanding the function of (cytosine-5) RNA methylation in RNA biology and in human disease.

overall_design: mRNA bisulfite sequencing of Tet1-/-,Tet2-/-,Tet3-/- embryonic stem cells were generated by deep sequencing, in triplicate, using Illumina

first: Carine

last: Legrand

first: Frank

last: Lyko


title: The microprotein Minion controls cell fusion and muscle formation

city: San Diego

first: John

middle: R

last: Walker

address: 10675 John Jay Hopkins

institute: Genomics Institute of the Novartis Research Foundation

summary: These findings establish minion as a novel microprotein required for muscle development, and define a two-component program for the induction of mammalian cell fusion.

overall_design: RNAseq of muscle samples before and after cardiotoxin injury

first: Qiao

last: Zhang

first: Ajay

last: Vashisht

first: Jason

last: O’Rourke

first: Stéphane

middle: Y

last: Corbel

first: Rita

last: Moran

first: Angelica

last: Romero

first: Loren

last: Miraglia

first: Jia

first: Eric

last: Durrant

first: Christian

last: Schmedt

first: Srinath

last: Sampath

first: Srihari


title: Neuroblastoma is composed of two super enhancer-associated differentiation states

city: Amsterdam

first: Richard

last: Volckmann

state: Noord-Holland

address: van der Boechorststraat 6a

department: Laboratory of Experimental Oncology and Radiobiology

country: Netherlands

institute: msterdam UMC and University of Amsterdam

summary: Intra-tumour heterogeneity is increasingly appreciated as a determinant of tumour recurrence. Several tumour types were recently found to include phenotypically divergent cell types, reflecting lineage development stages (1,2,3). Lineage identity has been proposed to ensue super-enhancer (SE)-associated transcription factor (TF) networks (4,5), but their role in intra-tumour heterogeneity is unknown. Neuroblastoma is a paediatric tumour of the adrenergic differentiation lineage. Here we show that most neuroblastoma tumors include two types of tumor cells with highly diverging gene expression profiles. The undifferentiated mesenchymal cells and more differentiated adrenergic cells can interconvert and may relate to normal  lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic neuroblastoma cells revealed a distinct, highly consistent super-enhancer landscape for each cell type. Two SE-associated TF networks emerged that potentially master each cell type. Accordingly, the mesenchymal TF PRRX1 could reprogram the SE- and mRNA-profiles of adrenergic cells towards a mesenchymal state. To assess the clinical relevance of this bi-phasic system, we investigated chemo-sensitivity of both cell types. Mesenchymal cells were more resistant in vitro and were enriched in post-therapy and relapsed neuroblastoma in patients. Intra-tumor heterogeneity in neuroblastoma is therefore structured according to distinct SE-associated transcriptional programs that mediate a dynamic bi-phasic structure.

overall_design: Cultured cells were fixed with 1% formaldehyde. Nuclei were isolated and the DNA was sheared to 200 to 300 bp fragments. Histon-bound DNA was precipitated using antibodies H3K27Ac (#4729, Abcam) and H3K4me3 (#04-745, Millipore).  De-crosslinked DNA was purified using Qiagen PCR purification kit (Qiagen) and quantified with Quant-IT Picogreen (Invitrogen). The DNA was used to generate sequencing libraries according to the manufactures procedure (Life Technologies): The DNA was end-polished, dA-tailed and adaptors with barcodes were ligated. The fragments were amplified (8 cycles) and quantified with a Bioanalyzer (Agilent). The libraries were prepped with the 5500W Flowchip v2 kit  (Life Technologies) and sequenced on the SOLiD Wildfire (Illumina) resulting in 50 bp reads. Alternatively, the libraries were sequenced using the HiSeq PE cluster kit v4 (Illumina) with the HiSeq2500 (Illumina) resulting in 125 bp reads.

first: L

last: Valentijn

first: J

last: Koster

first: D

last: Zwijnenburg

first: R

last: Versteeg


title: RNA-Seq profiling of brain in Mucopolysaccharidosis type II mouse model

city: Padova

first: Francesca

last: D'Avanzo

state: PD

address: c/o Pediatric Research Institute, Corso Stati Uniti, 4

department: Women’s and Children’s Health

institute: University of Padova

summary: Lysosomal Storage Disorders (LSDs) are a group of about 50 metabolic disorders, sharing the inability to degrade specific endolysosomal substrates. This results in failure of cellular functions in many organ systems including brain in most patients, which show a progressive neurodegeneration. In this study we performed an RNA-Seq analysis of two brain areas (cerebral cortex and midbrain/diencephalon/hippocampus) of the mouse model for Mucopolysaccharidosis type II, a neurodegenerative LSD.

overall_design: mRNA profiles of 9 month old wild type (WT) and Ids-ko (H) mice were generated by RNA-seq using AB SOLiD 3 Plus System

first: Marika

last: Salvalaio

first: Laura

last: Rigon

first: Alessandra

last: Zanetti

first: Michela

last: D'Angelo

first: Giorgio

last: Valle

first: Maurizio

last: Scarpa

first: Rosella

last: Tomanin


title: Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

last: Tuorto

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

overall_design: Refer to individual Series


title: CPK signaling orchestrates the plant nitrate regulatory network

first: Jen

last: Sheen

address: 185 Cambridge st. CPZN7620

department: Molecular Biology

institute: Massachusetts General Hospital

summary: Nitrate is the limiting nitrogen nutrient enabling photosynthetic plants to support the conversion of inorganic elements to organic biomass, which sustains all lives. Plants evolved multifaceted nitrate responses to modulate global gene expression, metabolism and developmental programs. However, primary nitrate signaling mechanisms remained elusive. Using an ultrasensitive Ca2+ biosensor, unique nitrate-induced Ca2+ signaling was illuminated in Arabidopsis cells and plants. Integrative functional genomic screens, chemical genetics, genome-wide transcript sequencing, and analyses of nitrate-associated traits uncovered the surprising roles of Ca2+ sensor protein kinases (CPKs) in orchestrating diverse primary and long-term nitrate responses. Nitrate specifies CPK signaling to reprogram transcriptome and govern N-assimilation, metabolism, transport, hormones, shoot growth, and root system architecture. CPKs may be targeted to enhance nitrogen-use-efficiency, reduce fertilizer demands, and alleviate ecosystem pollution.

overall_design: genome-wide survey of the wild type and icpk mutant in response to nitrate

first: Kun-hsiang

last: Liu

first: Yajie

last: Niu

first: Matthew

last: McCormack

first: Hoo

last: Chung


title: Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs [Whole-transcriptome bisulfite sequencing]

overall_design: Whole-transcriptome bisulfite sequencing of wild type and Dnmt2-/- embryonic stem cells were generated by deep sequencing, in triplicate, using Illumina

first: Carine

last: Legrand

first: Frank

last: Lyko


title: Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5

city: Beijing

first: xiaofeng

last: cao

address: West Lincui Road, Chaoyang District

department: State Key Laboratory of Plant Genomics

institute: Institute of Genetics and Developmental Biology

summary: In this study, we analyzed the pre-mRNA splicing status in wild-type Col, atprmt5 mutants, and two atprmt5 suppressors (m90 and s215). RNA-seq analyses revealed that m90 and s215 can partially rescue the pre-mRNA splicing defects in atprmt5 mutants. Further study showed that m90 and s215 were identified in the highly conserved pre-mRNA splicing factor 8 (AtPrp8) and  demonstrated that Prp19C/NTC complex failed to be assembled into U5 snRNP to form activated spliceosome in atprmt5 mutants, while restored in the suppressor. Our findings uncover a key molecular mechanism for AtPRMT5 in the regulation of pre-mRNA splicing.

overall_design: Investigate the pre-mRNA splicing in the suppressors of atprmt5 mutants

first: Xian

last: Deng

first: Tiancong

last: Lu

first: Lulu

last: Wang

first: Lianfeng

last: Gu

first: Chunyan

first: Xiangfeng

last: Kong

first: Xiaofeng

last: Cao


title: The histone variant H2A.Z promotes efficient co-transcriptional splicing in S. cerevisiae

status: Public on May 18 2017

city: Los Angeles

first: Stephen

last: Douglass

address: 5128 TLSB Terasaki Life Sciences Building, 610 Charles Young Drive East

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

summary: In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre-messenger RNA (pre-mRNA). Recent studies show the process of RNA-synthesis and RNA-processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited into chromatin by the SWR1-complex, is found near the 5’ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP, as H2A.Z shows extensive genetic interactions with U2 snRNP associated proteins, and RNA-seq reveals introns with non-consensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z’s role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled and unspliced RNAs are released. Together these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

overall_design: Transcriptomes of 11 different cell types in S. cerevisiae generated by sequencing of one to two independent preparations of rRNA-depleteted RNA libraries

first: Lauren

last: Neves

first: Roberto

last: Spreafico

first: Srivats

last: Venkataramanan

first: Tracy

last: Kress

last: Johnson


title: The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

city: Freising

first: Nils

last: Thieme

state: Bavaria

address: Hans-Carl-von-Carlowitz-Platz 2

department: Holzforschung München

institute: Technische Universität München

summary:
Background:
Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction.

Results:
The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of L-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA binding motif.

Conclusions:
PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.

The data here are associated with the following BioProjects: PRJNA256749, PRJNA349646, PRJNA349647, PRJNA349695, PRJNA349696, PRJNA349697, PRJNA349547, PRJNA349548, PRJNA349549, PRJNA349698, PRJNA349699, PRJNA349700

See the links on GSM entries for access to raw data at SRA.

overall_design: Data was gathered from carbon source transfer experiments; N. crassa WT and Δpdr-1 mutant were pre-grown for 16 hrs on sucrose, washed 3 times in media without carbon-source and then transferred to either sucrose (WT only), L-rhamnose, pectin or no carbon source, the samples were induced for additional 4 hrs; all samples were performed in biological triplicates except WT on pectin, which was performed in duplicates; sequencing was perfomed on the Illumina HiSeq2000

first: N

first: V

middle: W

last: Wu

first: A

last: Dietschmann

last: Salamov

first: M

first: J

last: Singan

first: I

middle: V

last: Grigoriev

last: Glass

first: C

last: Somerville

middle: P

last: Benz


title: Identification of gene expression patterns in spleen of 564+/- mice of different ages

city: Seoul

first: Chong-Su

last: Kim

address: Gwanak-ro 1, Gwanak-gu

department: Food and nutrition

institute: Seoul national university

summary: 564Igi mice homozygous for knockin alleles of 564 IgH and IgK expressed high levels of autoantibodies and exhibited a high incidence of mature B cell lymphomas. This study was designed to evaluate the gene expression profiles of 564Igi mice with different ages.

overall_design: 12 samples were tested from 4 groups of mice - 2 samples from one group and 3 to 4 samples from the other three groups. One sample per array

first: Dong-Mi

last: Shin

first: Shweta

last: Jain

first: Herbert

last: III Morse


title: Identification of differentially expressed genes in longissimus dorsi muscle between Wei and Yorkshire pigs using RNA-sequencing

status: Public on May 20 2017

city: Chuzhou

first: jingen

last: Xu

state: Anhui

address: No. 9 Donghua Road

department: College of Animal Science

institute: Anhui Science and Technology University

summary: Intramuscular fat (IMF) content is an important trait closely correlated with meat quality, which is highly variable among swine breeds from diverse genetic backgrounds. In order to elucidate the molecular mechanism underlying porcine meat quality, we adopted RNA-sequencing to detect transcriptome in the longissimus dorsi muscle of Wei pigs (a Chinese indigenous breed) and Yorkshire pigs (a Western lean-type breed) with different IMF content. A total of 717 differentially expressed genes (DEGs) were identified in our study, with 323 up-regulated and 394 down-regulated genes in Wei pigs compared with Yorkshire pigs. GO analysis showed that DEGs significantly related to muscle proliferation and development, lipid storage and catabolic, extracellular matrix structural constituent, and neutral amino acid transmembrane transporter activity. Pathway analysis revealed that DEGs associated with fatty acid metabolism, steroid biosynthesis, glycerophospholipid metabolism, protein digestion and absorption, and amino acid metabolism. Quantitative real time PCR confirmed the differential expression of 11 selected DEGs from both pig breeds. The results provide useful information to investigate the transcriptional profiling in skeletal muscle of different pig breeds with divergent phenotypes, and several DEGs can be taken as functional candidate genes for affecting pork quality.

overall_design: Transcriptome sequencing in longissimus dorsi muscle of Wei and Yorkshire pigs

first: Jingen

first: Shenghe

last: Li


title: Gene expression analysis of the effect of UV-B radiation in the growth zone of the maize leaf

city: Antwerpen

first: Gerrit

last: Beemster

state: Antwerpen

address: Groenenborgerlaan 171

department: Biology

country: Belgium

institute: University of Antwerp

summary: UV-B radiation affects leaf growth in a wide range of species. In this work, we demonstrate that UV-B levels present in solar radiation inhibits maize leaf growth without causing any other visible stress symptoms, including accumulation of DNA damage. We conducted kinematic analyses of cell division and expansion to understand the impact of UV-B radiation on these cellular processes. Our results demonstrate that the decrease in leaf growth is a consequence of a reduction in cell production, and a shortened growth zone (GZ) in UV-B irradiated leaves. To determine the molecular pathways involved in UV-B inhibition of leaf growth, we performed RNA sequencing on isolated GZ tissues of control and UV-B exposed plants. Our results show a link between the observed leaf growth inhibition and the expression of specific cell cycle and developmental genes, including Growth Regulating Factors (GRFs) and transcripts for proteins participating in different hormone pathways.

overall_design: Factorial design with two factors: Treatment (control vs UV-B) x Zone I (0-1cm from base of the leaf), 2 (1-2cm from base of the leaf) and 3 (2-3cm from base of the leaf), 3 replicates

first: Paula

last: Casati


title: DUSP9 is a dose-dependent, X-linked modulator of female DNA hypomethylation in mouse pluripotent stem cells

first: Konrad

last: Hochedlinger

address: 185 Cambridge St.

summary: Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X-chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. Notably, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.

overall_design: Transcriptional and epigenetic patterns were compared between genetically matched ESCs and EGCs were analysed.

first: Jiho

last: Choi

first: Kendell

last: Clement

first: Alexander

last: Meissner


title: Bromodomain protein BRD4 is a transcriptional repressor of autophagy and lysosomal function

city: Glasgow

first: Kevin

last: Ryan

address: Garscube Estate, Switchback Road

institute: Cancer Research UK Beatson Institute

summary: Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.

overall_design: RNA-Seq of KP-4 pancreatic adenocarcinoma cells transfected with control, BRD4 #1 or BRD4 #2 siRNA for 72hrs (n=3 independent sample preparations)

first: Jun-ichi

last: Sakamaki


title: Analysis of the glutathione-deficient lens transcriptome

city: Cincinnati

first: Mario

last: Medvedovic

address: 3223 Eden Av. ML 56

department: Department of Environmental Health

institute: University of Cincinnati

summary:
Purpose: To better understand the effects of glutathione (GSH)-deficiency on lens homeostasis and cataractogenesis.
Methods: The transcriptome of lens epithelia and fiber cells was obtained from C57BL/6 LEGSKO (lens GSH synthesis knockout) and buthionine sulfoximine (BSO)-treated LEGSKO mice and compared to C57BL/6 wild-type mice using RNA-Seq.
Results: RNA-Seq results were in excellent agreement with qPCR (correlation coefficients between 0.87-0.94 and p<5E-6 for a subset of 36 mRNAs). Of the 24,415 transcripts mapped to the mouse genome, 441 genes showed significantly modulated expression. Pathway analysis indicated major changes in EMT signaling, visual cycle, small molecule biochemistry, and lipid metabolism. GSH-deficient lenses showed upregulation of genes relating to detoxification, including Aldh1a1, Aldh3a1 (aldehyde dehydrogenases), Mt1, Mt2 (metallothioneins), Ces1g (carboxylesterase), and Slc14a1 (urea transporter UT-B). These proteins share substrate specificity with GSH or glutathione-S-transferase and may protect GSH-deficient lenses. Genes associated with canonical EMT pathways, including Wnt10a, Egf, and Syk, showed upregulation in lens epithelia samples. Severely GSH-deficient lens epithelia showed a broad downregulation of vision-related genes (including Cryge, Crygf, and Rho). The BSO-treated LEGSKO lens epithelia transcriptome has significant correlation (r=0.63,P<0.005) to that of lens epithelia undergoing EMT.
Conclusions: These results show that GSH depletion of the lens leads to expression of detoxifying genes and activation of EMT signaling, in addition to changes in transport systems and lipid homeostasis. These data give new insight into the adaptation and consequences of GSH-deficiency in the lens and suggest that supplementation of GSH or a precursor after cataract surgery could potentially reduce the incidence of EMT-mediated posterior subcapsular opacification.

overall_design: Lens epithelia and cortical fiber cells were isolated and only compared to samples of the same tissue type. Wild-type (control) mouse lens tissue was compared to LEGSKO (mildly glutathione deficient) lens tissue and buthionine sulfoxime (BSO)-treated LEGSKO (severely glutathione deficient) lens tissue.

first: Jeremy

last: Whitson

first: Xiang

last: Zhang

first: Jenny

last: Chen

first: Vincent

last: Monnier

first: Xingjun

last: Fan


title: Blocking promiscuous activation at cryptic promoters directs cell type–specific gene expression

city: Ithaca

first: Jongmin

last: Kim

state: NY

address: 930 Campus Road

department: Biomedical Sciences

institute: Cornell University

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

Abstract
To selectively express cell type–specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. Here, we show in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. Our results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation.


title: ChIP-Seq with Kumgang (Kmg), dMi-2, and Aly from wild-type and kmg KD Drosophila melanogaster testes

summary: Genome wide localization of Kumgang, dMi-2, and Aly in Drosophila melanogaster testes were evaluated by ChIP-Seq in wild-type and kmg knock down testes. / Title: Blocking promiscuous activation at cryptic promoters directs cell type–specific gene expression / Abstract:  To selectively express cell type–specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. Here, we show in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. Our results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation.

overall_design: 1) Kmg: Two replicates of ChIP of endogenous Kmg with specific antibody from wild-type testes. Two replicates of ChIP of Kmg from kmg knock down testes serve as negative controls / 2) dMi-2: Two replicates of ChIP of endogenous dMi-2 from wild-type testes and from kmg knock down testes / 3) Aly: Two replicates of ChIP of epitope(HA) tagged Aly from transgenic line bearing alyP-Aly-HA from wild-type for kmg and also from kmg KD testes. One ChIP of HA epitope from wild-type testes without Aly-HA transgene serves as a negative control.

first: Margaret

last: Fuller


title: Gene expression of fly testes with dMi-2, kumgang (CG5204) knock downs and wild-type heads by RNA-Seq

summary: The effect of different spermatocyte-specific loss of functions; kumgang (kmg or CG5204), dMi-2 in the gene expression in fly testes was assessed by RNA-Seq. Gene expression in wild-type heads were also measured to have a reference expression profile of 'somatic tissues'.  / Title: Blocking promiscuous activation at cryptic promoters directs cell type–specific gene expression / Abstract:  To selectively express cell type–specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. Here, we show in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. Our results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation.

overall_design: Gene expression profile of kumgang and dMi-2 knock down testes and corresponding sibling control testes (no Gal4 driver), and wild-type heads were generated in duplicate using Illumina NextSeq 500.


title: RNA binding by the histone methyltransferases Set1 and Set2

city: Portsmouth

first: Samuel

last: Robson

address: St Michael's Building

department: School of Pharmacy & Biomedical Science

institute: University of Portsmouth

summary: Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo. High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional post-transcriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

overall_design: RNA Seq expression profiling of the S. cerevisiae transcriptome was conducted to compared the expression of transcripts in wild-type W303 cells (WT) and W303 cells with set2::His3 knockout (SET2 KO). All conditions were run in triplicate.

first: Camille

last: Sayou

first: Gonzalo

last: Millán-Zambrano

first: Helena

last: Santos-Rosa

first: Elisabeth

last: Petfalski

first: Jonathon

last: Houseley

first: Tony

last: Kouzarides

first: David

last: Tollervey


title: Conversion of adult endothelium to immunocompetent haematopoietic stem cells

city: New York City

first: Balvir

last: Kunar

address: 1300 York Ave

department: Regenerative Medicine; Physiology, Biophysics, & Systems Biology

institute: Weill Cornell Medical College

summary: Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGF? and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

overall_design: Expression profiling by high throughput sequencing data; GPL17021 Illumina HiSeq 2500 (Mus musculus)

first: Raphael

last: Lis

first: Shahin

last: Rafii


title: The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retention

city: Dallas

first: Nicholas

middle: K

last: Conrad

state: TX

address: 6000 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

summary: Maintenance of the intracellular levels of the methyl donor S-adenosylmethionine (SAM) is essential for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of MAT2A, which encodes the only SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3´ UTR. Increasing METTL16 occupancy on the MAT2A 3´ UTR is sufficient to induce efficient splicing. We propose that under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which in turn promotes MAT2A splicing. Interestingly, human and S. pombe METTL16 methylate the U6 spliceosomal snRNA at a sequence identical to hp1. These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

overall_design: m6A-seq was performed on polyA-selected RNA from HEK293A-TOA cells from siCtrl or siMETTL16 knockdown conditions. 3 biological replicates were performed for each condition. Both the m6A-IP and the inputs are available for each sample.

first: Kathryn

last: Pendleton

first: Beibei

first: Kuanqing

last: Liu

first: Olga

last: Hunter

first: Yang

last: Xie

first: Benjamin

last: Tu


title: Evolutionary re-wiring of p63 regulatory landscape has both epigenetic and transcriptomic implications and is the underlying cause for epidermal differences between mouse and human [RNA-seq]

city: Buffalo

first: Satrajit

last: Sinha

address: 701 Ellicott Street

department: Department of Biochemistry

institute: State University of New York at Buffalo

summary: Gene expression analysis of  two different mouse keratinocytes using RNA-Seq

overall_design: RNA was collected and analyzed for two biological replicates each from two different mouse keratinocyte cell lines

first: Isha

last: Sethi

first: Christian

last: Gluck

first: Michael

last: Buck


title: Evolutionary re-wiring of p63 regulatory landscape has both epigenetic and transcriptomic implications and is the underlying cause for epidermal differences between mouse and human

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

overall_design: Refer to individual Series


title: Mesenchymal differentiation of neuroblastoma cell lines

first: Richard

last: Volckmann

address: van der Boechorststraat 6a