title: Next Generation Sequencing Facilitates Quantitative Analysis of Bone Marrow Derived Mast Cell Transcriptomes

city: shang hai

first: Qian

last: Zhang

address: xiangyin road 800

institute: Second military medical university

summary: Purpose:The purpose of this study is to detect activated or silenced genes during Tet2-deficient bone marrow derived mast cell (BMMC) and the control cells. Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods:Mouse BMMCs were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse IL-3 and SCF. BMMCs were stained to confirm the surface expression of FcɛRI and c-Kit. Cells with purity >97.5% were used for subsequent experiments. BMMCs RNA profiles were generated by deep sequencing,using Illumina. Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified hundreds of genes with significant mRNA variation between Tet2-deficient BMMC and the control cells.

overall_design: BMMC mRNA profiles of wild type (WT) and Tet2-/- mice were generated by deep sequencing.

first: Qicong

last: Shen

first: Xuetao

last: Cao


title: ChIP-seq analysis of H3K27ac, GATA1 and TAL1 in MYO1D hub vs non-hub enhancer knockout K562 cells

status: Public on Jan 27 2018

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

department: Pathology

country: USA

institute: St. Jude Children's Research Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Recent studies have highlighted super-enhancers (SEs) as important regulatory elements for gene expression, but their intrinsic properties remain incompletely characterized. Through an integrative analysis of Hi-C and ChIP-seq data, we find that a significant fraction of SEs are hierarchically organized, containing both hub and non-hub enhancers. Hub enhancers share similar histone marks with non-hub enhancers, but are distinctly associated with cohesin and CTCF binding sites and disease-associated genetic variants. Genetic ablation of hub enhancers results in profound defects in gene activation and local chromatin landscape. As such, hub enhancers are the major constituents responsible for SE functional and structural organization.

overall_design:
ChIP-seq was performed to determine the chromatin occupancy of H3K27ac, GATA1 and TAL1 in MYO1D hub or non-hub enhancer knockout K562 cells.

MYO1D Non-hub enhancer deletion: chr17:31220757+31222033 (1277 bp)
MYO1D Hub enhancer deletion: chr17:31236528+31240709 (4182 bp)

first: Kailong

last: Li

first: Yuannyu


title: Weighted gene coexpression analysis of RNA-seq data from 65d juvenile Area X and adjacent non-song ventral striatopallidum (VSP).

status: Public on Jan 25 2018

city: Los Angeles

first: Zachary

middle: Daniel

last: Burkett

state: CA

address: 610 Charles E Young Drive East Room 1045

department: Integrative Biology & Physiology

institute: University of California, Los Angeles

type: Expression profiling by high throughput sequencing

summary: Human speech is one of the few examples of vocal learning among mammals, yet ~half of avian species exhibit this ability. Its genetic basis is unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. Here we manipulated FoxP2 isoforms in Area X during a critical period for song development, delineating, for the first time, unique contributions of each to vocal learning. We used weighted gene coexpression network analysis of RNA-seq data to construct transcriptional profiles and found gene modules correlated to singing, learning, or vocal variability. The juvenile song modules were preserved adults, whereas the learning modules were not. The learning modules were preserved in the striatopallidum adjacent to Area X whereas the song modules were not. The confluence of learning and singing coexpression in juvenile, but not adult, Area X may underscore molecular processes that drive vocal learning in zebra finches and, by analogy, humans.

overall_design: 19 samples each from Area X and VSP for birds injected in Area X with AAV1 to overexpress GFP, full length FoxP2, or truncated FoxP2. Biological replicates but not technical replicates are included.

middle: D

first: Nancy

middle: F

last: Day

first: Todd

middle: H

last: Kimball

first: Caitlin

middle: M

last: Aamodt

first: Jonathan

middle: B

last: Heston

first: Austin

middle: T

last: Hilliard

first: Xinshu

last: Xiao

first: Stephanie

middle: A

last: White


title: Distinct 5-methylcytosine profiles in poly(A)RNA from mouse bone marrow derived mast cells

country: China

type: Methylation profiling by high throughput sequencing

summary: Purpose: To transcriptome widely reveal 5-mC regulation by Tet2, we performed bisulfite-sequencing of mRNAs from Tet2-deficient BMMCs and the control cells.Methods: Mouse BMMCs were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse IL-3 and SCF. BMMCs were stained to confirm the surface expression of FcɛRI and c-Kit. Cells with purity >97.5% were used for RNA isolation. Results: We carried out an unbiased global analysis of m5C in poly(A)RNA of mouse bone marrow derived mast cells (BMMCs).We show that there were indeed much more mCs in Tet2-deficient group compared with the control, more than half of which located in genic region. Furthermore, we observed that although much less mCs distributed in the three elements of exon regions, more mCs located in 3`-UTR for Tet2-deficient group compared with the control.

overall_design: RNA was isolated from Tet2 deficient bone marrow derived mast cells (BMMC) and control cells and RNA-Bisulfite sequencing was performed. We performed two biological replicates and each contain two technical replicates


title: Cross-linking and immunoprecipitation (CLIP) analysis for identifying binding sites of Tet2 in bone marrow derived mast cell

summary: To transcriptome-widely identify the mRNA targets of Tet2 and investigate the immunological function of Tet2 at RNA regulation level in vivo, we performed CLIP-seq of Tet2 in BMMCs. We performed three bio-replicates of CLIP-seq for getting potential Tet2-binding mRNAs.Using data of normalized tag numbers in the common peaks, we found that biological replicates correlated well with each other.More than 60% of the peaks located in genic regions, which were preferentially enriched in coding sequencing (CDS) among mature mRNA elements.

overall_design: Systematically identify Tet2-binding RNAs in biological triplicate.


title: The Cytoplasmic mRNA Decay Landscape of Arabidopsis Seedlings [small RNA-seq]

status: Public on Jan 26 2018

city: Salt Lake City

first: Leslie

middle: Ellen

last: Sieburth

state: UT

address: 257 South 1400 East

department: Biology

institute: University of Utah

type: Non-coding RNA profiling by high throughput sequencing

summary: Cytoplasmic mRNA decay occurs through several pathways, but the contributions of these decay pathways to the degradation of specific mRNAs, and interactions between the pathways, are not well understood.  We carried out a genome-wide analysis of mRNA decay rates using wild-type Arabidopsis and mutants with defects in mRNA decapping and SOV/DIS3L2.  Decay rates and contributions of decapping and SOV to decay were estimated for 18,674 mRNAs using maximum likelihood modeling. Most mRNAs decayed by multiple pathways, few mRNAs degraded exclusively by mRNA decapping or SOV, and specific codon usage was linked to decay rates. Unexpected faster decay of transcripts in some genotypes was found to be independent of siRNAs; instead the data suggested an RNA buffering-like phenomenon in Arabidopsis, and that VCS (decapping) is essential for both this process and the decay of very unstable mRNAs.

overall_design: Small RNA abundance measured in 5 day-old seedlings to compare to same time 0 decay rates in genome-wide decay dataset.

middle: E

first: Malia

middle: J

last: Deshotel

first: Reed

last: Sorenson

first: Katrina

last: Johnson

first: Frederick

middle: R

last: Adler


title: Gene induction by the USP6 oncogene in response to interferon

status: Public on Jan 24 2018

city: Philadelphia

first: Ian

last: Henrich

state: PA

address: 803D Abramason Research Center, 3615 Civic Center Blvd

institute: University of Pennsylvania

summary: We examined the role of the USP6 oncogene in the patient-derived immortalized ewing sarcoma cell line RD-ES. RNA-sequncing revealed that USP6 induces numerous genes that overlap with genes induced by interferon treatment. When USP6-expressing cells were treated with IFN, many of the overlapping genes were synergistically upregulated, indicating an overlap in signaling pathways.

overall_design: Examine the effects of USP6 and/or IFN on gene induction in RD-ES cells

first: Margaret

last: Chou


title: Endothelial gene expression analysis

city: Frankfurt am Main

first: Philipp

last: Neumann

state: Hessen

address: Theodor-Stern-Kai 7

country: Germany

institute: Institute for Cardiovascular Regeneration

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

overall_design: Refer to individual Series


title: Next Generation Sequencing Facilitates Quantitative Analysis of either Pkd1f/f, Pkd1f/f:HoxB7-Cre mice or Pkd2f/f, Pkd2f/f:HoxB7-Cre mice

city: Seoul

first: Jong Hoon

last: Park

address: Cheongpa-ro 47-gil 100, Yongsan-gu,

country: South Korea

institute: Sookmyung Women's university


title: The Cytoplasmic mRNA Decay Landscape of Arabidopsis Seedlings [RNA-seq]

overall_design: RNA abundance measurements at 0, 7.5, 15, 30, 60, 120, 240, 480 min after treatment with 1 mM cordycepin (transcription inhibitor) in 4 decay pathway genotypes  (WT, vcs, sov, vcs sov; 4 bioreps each) to model effects on decay rate.


title: Small RNA-seq of human HEK293 cells overexpressing mmu-miR-1948 primary sequence

city: Boston

first: David

middle: J.

last: Waxman

state: MA

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

summary: Ectopic expression of mmu-miR-1948 primary sequence in HEK293 cells was followed by detection of mature mmu-miR-1948 by small RNA-seq to verify the authenticity of miR-1948

overall_design: The mmu-miR-1948 hairpin sequence plus the surrounding ~200 bp sequence were overexpressed in HEK293 cells by adenovirus infection.  Small RNA-seq was performed on the infected HEK293 cells to determine the presence of processed miR-1948 mature microRNAs.

first: Pengying

last: Hao


title: Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures

city: Cambridge

first: Federico

last: Comoglio

address: Hills Road

country: United Kingdom

institute: University of Cambridge

summary: Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis-regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, homotypic TPO-responsive enhancers are spatially clustered and engage in preferential intra- and inter-TAD interactions that are only moderately perturbed by TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that activated and repressed cis-regulatory elements share a remarkably similar DNA sequence composition and that in vivo transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

overall_design: Subcellular RNA-seq, ChIP-seq, Hi-C and Promoter Capture Hi-C in HPC7 cells and two biological conditions.

first: Hyun

first: Stefan

last: Schoenfelder

first: Iros

last: Barozzi

first: Daniel

last: Bode

first: Peter

last: Fraser

first: Anthony

last: Green


title: Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis [TCRseq]

city: La Jolla

first: Pandurangan

last: Vijayanand

state: California

address: 9420 Athena Circle

department: Division of Vaccine Discovery

institute: La Jolla Institute for Allergy and Immunology

summary: CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, heterogeneity and clonal diversity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile and clonality in humans. The single-cell differential gene expression analysis, revealed a spectrum of known transcripts, including several linked to cytotoxic and co-stimulatory function, and transcripts of unknown cytotoxicity-related function that are expressed at higher levels in the TEMRA subset, which is highly enriched for CD4-CTLs, compared to cells in the central and effector memory subsets (TCM, TEM). Simultaneous T cells antigen receptor (TCR) analysis in single-cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared to TCM and TEM cells and that the majority of CD4-TEMRA were dengue virus (DENV)-specific in subjects with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across subjects, with four distinct clusters identified by the single-cell analysis. Most importantly, we identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and thus could provide insights into the mechanisms that may be utilized to generate durable and effective CD4-CTL immunity.

overall_design: TCR analysis of  purified populations of human CD4 memory cell subsets by TCR-sequencing.

first: Veena

middle: S

last: Patil

first: Ariel

last: Madrigal

first: Benjamin

last: Schmiedel

first: James

last: Clarke

first: Patrick

last: O'Rourke

first: Eva

last: Harris

first: Aruna

last: de Silva

first: Bjoern

last: Peters

first: Grégory

last: Seumois

first: Daniela

last: Weiskopf

first: Alessandro

last: Sette


title: Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis


title: Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis [bulk RNA-seq]

overall_design: Transcriptome  analysis of  purified populations of human CD4 memory cell subsets by bulk RNA-sequencing.


title: Analysis of transcriptional regulation by Myt1 and Myt1l

city: Charlotesville

first: David

last: Wotton

department: Biochemistry and Molecular Genetics

institute: University of Virginia

summary: We report the changes in gene expression in U87 glioblastoma cells with re-introduced Myt1 or Myt1l.

overall_design: U87 cells were stably infected with lentiviral vectors endoding Myt1, Myt1l, or with a control empty vector. We analyzed three replicate cultures for each condition by RNA-seq.

first: Agata

last: Lemiesz


title: Genome wide identification of gene sets involved in the regulation of hypothalamic pubertal development

city: Beaverton

first: Hollis

last: Wright

state: OR

address: 505 NW 185th Avenue

department: DIvision of Neuroscience

institute: ONPRC/OHSU

summary: Gene expression analysis of hypothalami from female animals at different juvenile developmental reproductive stages. Results provide insight into the role of the hypothalamus in controlling the onset of puberty.

overall_design: SD rats were housed (8/cage) in a controlled environment and euthanized at different ages (PND=7, PND=14, Early Juvenile: 21 days, Late Juvenile: 28 days, Late Proestus (the day of first ovulation): 30-33 days. Rats were anesthetized and brains were rapidly removed. The medial basal hypothalamus (MBH) was dissected away from the rest of the brain and flash frozen. Total RNA was isolated from each sample using Qiagen's RNeasy Mini Kit (Valencia, CA). Samples were bioanalyzed on a RNA 6000 Nano chip kit to check for integrity and concentration before sending it to OHSU's Massively Parallel Sequencing Shared Resource for library preparation and sequencing.

first: Alejandro

last: Lomniczi

first: Carlos

last: Toro

first: Sergio

last: Ojeda


title: Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor

city: San Diego

first: Matthew

last: Janes

address: 3033 Science Park Rd, Suite 220

institute: Wellspring Biosciences

summary: We report RNAseq gene expression data following ARS-1620 treatment and shKRAS expressing cells (NCI-H358).  We also compare gene expression changes following treatment with ARS-1620 or trametinib in NCI-H358, LU65 (KRAS-G12C+), and A549 (KRAS-G12S+) cells.  Additionally we report a time course (4, 24, 48hr) of ARS-1620 and trametinib treated NCI-H358 cells.

overall_design: Examination of whole transcriptome RNAseq data from 3 separate experimental designs: 1. H358 cells, treated with ARS-1620 & DMSO (24hr) and transduced with shRNA control & KRAS (24hr post infection), 2. H358, LU65, and A549 cells treated with DMSO, ARS-1620, or trametinib (for 24hr), and 3. H358 cells treated as time course of ARS-1620 (1uM) or trametinib (50nM)


title: Transcriptional and chromatin changes accompanying de novo formation of transgenic piRNA clusters

city: Moscow

first: Sergei

last: Ryazansky

address: Kurchatov sq., 2

department: Department of Molecular Genetics of Cell

country: Russia

institute: Institute of Molecular Genetics

summary: Expression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a Heterochromatin Protein 1 (HP1) homolog. Previously, we have shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. We noted that identical transgenes located in different genomic sites vary considerably in piRNA production and classified them as “strong” and “weak” piRNA clusters. Here, we investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. We found that the formation of a transgenic piRNA cluster is accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the “empty” sites before transgene insertion. Finally, we have shown that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an “all– or– nothing” model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production.

overall_design: This study aims at characterizing the novel piRNA clusters formed at the site of transgene integration by using smallRNA-seq and GRO-seq in adult ovaries of transgenic Drosophila strains.

first: Natalia

last: Akulenko

first: Alla

last: Kalmykova


title: Total RNA deep sequencing (ribosomal depleted) of human umbilical vein endothelial cells exposed to hypoxia (0.2%) for 12h and 24h or kept under normoxic conditions.

first: Philipp

last: Neumann

summary: Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is up-regulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-?2-induced endothelial-mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function.

overall_design: Human umbilical vein endothelial cell ribo-minus RNA deep sequencing. We used two replicate samples per 12h and 24h of normoxic and hypoxic conditions.

first: Dimmeler

last: Stefanie


title: Ribothrypsis, a novel process of canonical mRNA decay, mediates ribosome-phased mRNA endonucleolysis.

status: Public on Jan 23 2018

city: Baltimore

first: Manolis

last: Maragkakis

state: MD

address: 251 Bayview Blvd

institute: National Institute on Aging

summary: By developing Akron-Seq, a novel approach that captures native 3' and 5' ends of capped and polyadenylated RNAs respectively, we show that canonical human mRNAs are subject to repeated, cotranslational, ribosome-phased, endonucleolytic cuts in a process that we term ribothrypsis.

overall_design: Capturing native 3' ends of capped and 5' ends of polyadenylated mRNAs

first: Fadia

last: Ibrahim

first: Panagiotis

last: Alexiou

first: Zissimos

last: Mourelatos


title: Hemicastration induced spermatogenesis related DNA methylation and gene expression changes in mice testis

city: chengdu

first: Yixin

last: Wang

state: sichuan

address: 211 huimin street

department: Institute of Animal Genetics and Breeding, College of Animal Science and Technology

institute: Sichuan Agricultural University


summary: Hemicastration is unilateral orchiectomy to remove an injured testis, and may influence spermatogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we monitored hemicastrated mice and found no significant difference of growth or testosterone (P > 0.05) compared with control. The genome-wide DNA methylation pattern of remaining testis were investigated by RRBS. Substantial genes harbored differentially methylated regions in gene bodies, and were enriched in process of protein binding and cell adhesion. Moreover, RNA-seq results indicated that 46 DEGs involved in meiotic cell cycle, synaptonemal complex assembly and spermatogenesis were upregulated in the hemicastration group, while 197 DEGs were downregulated, which were related to arachidonic acid metabolism. Integrative analysis revealed that PSMC3IP, which encodes a protein crucial for homologous recombination in spermatocytes,  exhibited promoter hypomethylation and higher expression level in hemicastrated mice. Our results demonstrated that hemicastration-induced compensatory response maintained normal growth and fertility in mice.

overall_design: A total of six right testis of healthy male KM mice with body maturation were used in this study: three individuals were performed prepubertal hemicastration to remove left testis; At the same time, three counterparts were performed shame surgery.


title: Comparative Transcriptomic Profiling of Normal-Appearing and Scarred Areas of the Lungs Reveals Pathobiological Clues to Idiopathic Pulmonary Fibrosis

middle: P

last: Atamas

address: 10 S. Pine St., MSTF 834

department: Department of Medicine

institute: University of Maryland School of Medicine

summary:
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with overtly scarred peripheral and basilar lung regions and macroscopically unaffected central lung areas.
OBJECTIVES: To gain better insight into IPF pathobiology by comparing transcriptomic profiles of normal-appearing and scarred regions of IPF lung.
METHODS: Lung tissue samples from macroscopically unaffected (normal-appearing, IPFn) and scarred (IPFs) regions of explanted IPF lungs were analyzed by RNASeq and compared with healthy control (HC) lung tissues. RT-qPCR and immunohistochemistry were used to confirm selected findings.
MEASUREMENTS AND RESULTS: Numerous previously reported IPF-associated gene expression disturbances as well as additional differentially expressed mRNAs were observed. There were profound transcriptomic changes in IPFn compared with HC tissues, which included elevated expression of extracellular matrix-, immunity- and inflammation-related mRNAs. The magnitude and statistical significance of these changes were comparable or greater than those in the IPFs-to-HC comparison. When directly compared with IPFn, IPFs tissues demonstrated elevated expression of epithelial mucociliary mRNAs. Compared with HC, both IPFn and IPFs tissues demonstrated reduced expression of mRNAs related to solute carrier membrane transport and metabolic processes. Primary fibroblast cultures from IPFn and IPFs tissues were transcriptomically identical.
CONCLUSIONS: Macroscopically normal-appearing IPF tissues demonstrate profound disease activity and substantially similar transcriptomic profiles to scarred areas. Differences between these tissues are due to cell types other than fibroblasts and notably include enhanced expression of mucociliary genes in scarred areas. Deranged epithelial homeostasis or possibly non-transcriptomic factors may thus explain the marked architectural differences between normal-appearing and terminally scarred lung in end-stage IPF.

overall_design: RNASeq of 26 lung tissue samples from patients with IPF, including affected and unaffected areas of the lung, and from healthy controls


title: Genomic occupancy of the INO80 complex [II]

city: Chapel Hill

first: Jesse

last: Raab

address: 120 Mason Farm Rd

department: Genetics

institute: University of North Carolina Chapel Hill

summary: We mapped 4 subunits of the INO80 complex using ChIP-seq in HepG2 liver cancer cells. We found a subclass of sites occupied by the INO80 ATPase subunit, but not by any accessory subunits that we call 'Non-canonical' INO80 sites. These sites are characterized by repressed chromatin.

overall_design:
ChIP-seq was performed for INO80 subunits in HepG2 cells and was processed for ChIP as previously described (Raab et al 2015), followed by high throughput sequencing.
Reanalysis of GSE97411 Samples.

first: J

first: T

last: Magnuson


title: Transcriptome profiling of A2M treated A549 Cell Line Samples

city: Leipzig

first: Ronny

last: Amberg

state: Saxony

address: Talstrasse 33

department: Faculty of Life Sciences

institute: University of Leipzig

summary: The goal of the study was to identify changes in transcriptome expressions upon treatment of A549 cell line samples with activated A2M.

overall_design: Sixteen A549 cell cultures divided into eight pairs were prepared. Each pair consisting of one A2M* treated and one untreated culture. Five samples could not be adequately quantified and subsequently needed to be excluded from downstream sequencing.

first: Rene

last: Thieme

first: Susanne

last: Kurz

first: Marco

last: Groth

first: Heinz-Georg

last: Jahnke

last: Pieroh

first: Lars-Christian

last: Horn

first: Marlen

last: Kolb

first: Klaus

last: Huse

first: Matthias

last: Platzer

first: Daniela

last: Volke

first: Faramarz

last: Dehghani

first: Anton

last: Buzdin

first: Kathrin

last: Engel

first: Andea

last: Robitzki

first: Ralf

last: Hoffmann

first: Ines

last: Gockel

first: Gerd

last: Birkenmeier


title: Id proteins suppress E2A-driven iNKT cell development prior to TCR selection [RNA-seq_new]

city: Durham

first: Yuan

last: Zhuang

state: NC

address: 207 Research Drive

department: Immunology

institute: Duke University

summary: Id proteins have been shown to promote the differentiation of conventional αβ and γδT cells, and to suppress the expansion of invariant Natural Killer T (iNKT) cells and innate-like γδNKT within their respective cell lineages. However, it remains to be determined whether Id proteins regulate lineage specification in developing T cells that give rise to these distinct cell fates. Here we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical for the iNKT cell lineage prior to TCR expression. Lack of Id proteins also promotes a biased TCR rearrangement in favor of iNKT cell fate prior to selection at the CD4+CD8+ double positive (DP) stage. Enhanced iNKT development in Id3-deficient mice lacking γδNKT cells suggests that Id3 regulates the lineage competition between these populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT and γδNKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a large population of iNKT cells and a unique innate-like DP population, despite the block in conventional αβ T cell development. The transcriptional profile of these unique DP cells reflects enrichment of innate-like signature genes, including PLZF (Zbtb16) and Granzyme A (Gzma). Results from these genetic models and genome-wide analyses suggest that Id proteins suppress E2A-driven innate-like T cell programs prior to TCR selection to enforce predominance of conventional T cells.

overall_design:
The RNA-Seq experiment included WT DP, pTαKO DP, L-DKO DP and L-DKO  pTαKO (abbreviated as LP) DP cells (where L-DKO refers to mice deficient in both Id2/Id3). Each replicate represents cells from a single mouse. One pTαKO DP (#1) sample was removed from analysis due to low quality of sequencing.
All mice were B6/129 hybrids and littermates.

first: Sumedha

last: Roy

first: Amanda

last: J. Moore

first: Cassandra

last: Love

first: Anupama

last: Reddy

first: Deepthi

last: Rajagopalan

first: Sandeep

last: Dave

first: Leping

last: Li

first: Cornelis

last: Murre


title: Identification of the TXNIP IRES and characterization of the impact of regulatory IRES trans-acting factors

city: Greifswald

first: Stefan

last: Simm

address: Walter-Rathenau-Straße 48

department: Bioinformatics

institute: University Medicine Greifswald

summary: Translation is a tightly regulated and is predominantly controlled at the level of its initiation. Initiation occurs in a cap-dependent manner. Under stress conditions when cap-dependent translation is hampered, internal ribosome entry sites (IRESes) allow for cap-independent translation of certain mRNAs. IRES-dependent translation is commonly regulated by RNA-interacting proteins, known as IRES trans-acting factors (ITAFs). In the present study, we searched for new IRESes by identifying 5’ untranslated regions (UTRs) bound by the ITAF hnRNPA1. Using a PAR-iCLIP approach, we found the mRNA of thioredoxin interacting protein (TXNIP) bound by hnRNPA1. Upon verification of an IRES element within the 5’UTR of TXNIP, we determined additional interacting proteins, which predominantly appeared to interact with the IRES-regulatory second half of the 5’UTR. Amongst these PTB, FBP3, and GEMIN5 emerged as functional ITAFs. Finally, we found that the TXNIP IRES-inhibitory effect of PTB was dominant over the activating effect of FBP3, while it succumbed to the stimulatory function of GEMIN5. In summary, we identified and characterized a novel IRES within the 5’UTR of TXNIP, which is regulated by the ITAFs PTB, FBP3, and GEMIN5.

overall_design: 1 Sample examined

first: Sebastian

last: Lampe

first: Michael

last: Kunze

first: Anica

last: Scholz

first: Thilo

last: Brauß

first: Sofia

last: Winslow

first: Mario

last: Keller

first: Juliane

last: Heidler

first: Ilka

last: Wittig

first: Bernhard

last: Brüne

first: Tobias

last: Schmid


title: Tn-Seq and RNA-seq of Benzalkonium Chloride Treated and Untreated Acinetobacter baumannii

city: Austin

first: Bryan

last: Davies

state: TX

address: 2506 Speedway

institute: University of Texas at Austin

overall_design: Refer to individual Series


title: Tn-Seq of Benzalkonium Chloride Treated and Untreated Acinetobacter baumannii

summary: The goal of this Tn-Seq study was to determine important determinants of Acinetobacter baumannii tolerance of sub-MIC concentrations of benzalkonium chloride.  This Tn-seq data was then utilized to aide in the determination of the sub-MIC mechanism of action for benzalkonium chloride.

overall_design: Two control samples and two treated samples were used

middle: W

first: Gregory

last: Knauf


title: NR4A1 and NR4A3 Restrict HSC Proliferation via Reciprocal Regulation of C/EBPα and Inflammatory Signaling

city: Houston

first: Pablo

middle: Riera

last: Freire

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: NR4A nuclear receptors are known tumor suppressors of acute myeloid leukemia and are essential regulators of hematopoietic stem cell (HSC) quiescence. To study the transcriptional consequences of acute NR4A1/3 codepletion in HSCs, we used an Nr4a1/3 conditional knockout mouse model and performed a global transcriptome profiling using RNA-Seq in sorted HSCs four days after NR4A1/3 codepletion.

overall_design: CDKO mice (Nr4a1 fl/fl; Nr4a3-/-; Rosa26-ERt2) and control mice (Nr4a1 fl/fl; Nr4a3-/-) were treated with 4 daily tamoxifen injections (120 mg/Kg) to incduce Nr4a1 excision and hematopoietic stem cells were sorted on day 5 for RNA-Seq analysis. Each sample consists in a pool of 3-5 mice.

first: Orla

last: Conneely


title: DNA methylation analysisof KRAS mutated colorectal tumors and adjacent tissues from cancer patinets and KRAS mutated Aberrant Crypt Foci and matching normal crypts from healthy individuals  by using RRBS

city: Duarte

first: Xiwei

last: Wu

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

institute: City of Hope National Medical Center

summary: Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study we have combined laser-capture microdissection (LCM) with reduced representation bisulfite sequencing (RRBS) to identify cancer associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology (GO) analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2 (PRC2).Consistent with their role in the earliest stages of colonic neoplasia, 75% of the methylation changes identified in ACF were also present in the CRC samples. Together, these data demonstrate that DNA methylation changes, including significant hypomethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

overall_design: RRBS of 10 pairs primary tumor/matching normal colon tissue; RRBS of 10 pairs Aberrant Crypt Foci/matching normal crypt.

first: Maria

last: Hahn

first: Matthew

last: Hanley

first: Arthur

middle: X

last: Li

first: Jianan

last: Lin

first: Jinhui

last: Wang

first: Audrey

last: Choi

first: Zhengqing

last: Ouyang

first: Yuman

last: Fong

first: Gerd

last: Pfeifer

first: Thomas

last: Deversj

first: Daniel

last: Rosenberg


title: Defining the early steps of cardiovascularlineage segregation by single cell RNA-seq

status: Public on Jan 28 2018

first: Evangelia

last: Diamanti

address: Wellcome Trust / MRC Building, Hills Road

department: Haematology

summary: Mouse heart development arises fromMesp1 expressing cardiovascular progenitors that are specified at the early stage of gastrulation. Lineage tracing and clonal analysisof Mesp1 progenitors revealed that heart development arises from distinct populations of Mesp1+ cardiovascular progenitors (CPs) expressing Mesp1 at different time points during gastrulation that contribute to different heart regions and different cardiovascular lineages. However, it remains unclear what are the molecular mechanisms that control the early regional and lineage segregation of Mesp1 CPs. Here, we performedsingle cell RNA-sequencing of FACS isolated Mesp1 CPs in WT and Mesp1 null embryos at different times to define the cellular and molecular heterogeneity of the Mesp1CPs, identify the role of Mesp1 in regulating cellular heterogeneity and uncover the mechanisms associated with lineage and regional segregation during the early stages of gastrulation.We showed that Mesp1 CPs isolated at E6.75 and E7.25 are molecularly distinct and make the continuum betweenepiblast and later mesodermal cells including hematopoietic progenitors.  Single cell transcriptome of Mesp1 deficient CPsshowed that Mesp1 is required for the exitof thepluripotent state and the induction of the cardiovascular gene expression program in vivo.Using dimensional reduction analysis, we identified distinct populations of Mesp1 CPs that correspond to progenitors committed to different celllineages and regions of the heart, identifying the molecular features associated with the early lineage and regional segregation. Notch1CREER lineage tracing, a marker preferentially expressed by one of the different Mesp1CP subpopulations, during the early stage of gastrulation marked almost exclusively ECs, demonstrating theexistence of an early Mesp1 subpopulation committed to the EC fate.This study uncoversthe cellular and molecular heterogeneity associated with early lineage restriction and regional segregation of the heart at the early stage of gastrulation.

overall_design: Single cell RNA sequencing of gastrulation E6.75 and E7.25 Mesp1+ cells

first: Fabienne

last: Lescroart

first: Xiaonan

first: Xionghui

first: Souhir

last: Gargouri

first: Victoria

last: Moignard

first: Christine

last: Dubois

first: Catherine

last: Paulissen

first: Berthold

last: Göttgens

first: Cédric

last: Blanpain


title: Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia

first: Xiwei

last: Wu

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


title: Stem cell intrinsic, Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors

city: Ashburn

first: Ken

last: Sugino

state: VA

address: 19700 Helix Dr

institute: Janelia Research Campus

summary: One neural stem cell can produce multiple transiently amplifying, intermediate neural progenitors (INP), which collectively yield diverse neuronal types. It is unclear if and how serially derived INPs contribute to neuron fate diversification. Drosophila type II neuroblasts, like mammalian neural stem cells, deposit neurons and also glia through INPs. The consecutively born INPs in a given lineage produce morphologically distinct progeny, presumably due to their inheritance of different temporal factors from the INP-producing progenitor. To uncover the underlying temporal fating mechanisms, we profiled type II neuroblasts' transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons/glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Further, we reveal that the transcription factor, Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts apparently locked in their beginning Imp/Syp levels can still yield progeny with a small range of early fates. We propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.

overall_design: Examination of one cell type (type II neuroblast) under 4 different time points (24hr, 36hr, 50hr, and 84hr after larva hatch).

first: Qingzhong

last: Ren

first: Ching-Po

last: Yang

first: Zhiyong

last: Liu

first: Tzumin

last: Lee


title: Hemicastration induced spermatogenesis-related DNA methylation and gene expression changes in mice testis

first: Yixin

last: Wang

overall_design: A total of four right testis of healthy male KM mice with body maturation were used in this study: two individuals were performed prepubertal hemicastration to remove left testis; At the same time, two counterparts were performed sham surgery.


title: Smed-hippo(RNAi) RNA-Seq

city: Barcelona

first: Gustavo

last: Rodriguez-Esteban

state: Catalonia

address: C. Baldiri Reixac, 4; Barcelona Science Park

department: Development

country: Spain

institute: Centro Nacional de Análisis Genómico

summary: The Hippo pathway plays a key role in regulating cell turnover in adult tissues, and abnormalities are consistently associated with human cancers. Initially related with the control of cell proliferation and death, hippo inhibition is commonly linked to an expansion of stem cells and progenitors, which leads to larger organs and tumors. To understand the mechanism through which Hippo directs cell renewal and promotes stemness, we studied its function in planarians, a stem cell-based organism that allows analysis of the complex cellular events underlying tissue renewal in the whole organism. We show that hippo inhibition in planarians decreases cell death, produces cell cycle arrest, and promotes the dedifferentiation of post-mitotic cells. Thus, hippo (RNAi) planarians have extensive undifferentiated areas and overgrowths with no effects on size or cell number. We propose an essential role for hippo in restricting cell plasticity and ensuring genomic stability, and thus in preventing tumoral transformation.

overall_design: Transcriptomic analysis on Smed-hippo(RNAi) animals after one, three and four weeks of inhibition. Three replicates were generated per condition from pools of animals.

last: Rodríguez-Esteban

first: Nídia

last: de Sousa

first: Teresa

last: Adell

first: Emili

last: Saló


title: Mnemiopsis Embryonic Developmental Timecourse

city: Kansas City

first: Alejandro

last: Sánchez Alvarado

state: Missouri

address: 1000 E 50th Street

institute: The Stowers Institute for Medical Research

summary: A time series from 1 hour to 9 hours post-fertilization (hpf) was collected for RNA-seq by students of the MBL Embryology Course to understand the shifts in gene expression occuring in the early embryo of Mnemiopsis leidyi.

overall_design: Multiple biological replicates were collected for timepoints 1, 2, 3, 4, 5, 6, 7, 8, and 9 hpf.

first: Antje

last: Fischer

first: Joel

last: Smith

first: Kirsten

last: Gotting

first: Eric

last: Ross


title: Coding and noncoding transcriptome sequencing of KRAS mutated colorectal tumors and adjacent tissues from cancer patients and KRAS mutated Aberrant Crypt Foci and matching normal crypts from healthy individuals [RNA-Seq]

last: Wu

overall_design: 10 pairs primary tumor/matching normal colon tissue; 5 pairs Aberrant Crypt Foci/matching normal crypt. 30 samples in total. Two technical replicates of the library preparation for sample 165S (165S1 and 165S2)

first: Maria

last: Hahn

first: Matthew

last: Hanley

first: Arthur

last: Li

first: Jianan

last: Lin

first: Jinhui

first: Audrey

last: Choi

first: Zhengqing

last: Ouyang

first: Yuman

last: Fong

first: Gerd

last: Pfeifer

first: Thomas

last: Deversj

first: Daniel

last: Rosenberg


title: Che-1 is targeted by c-Myc to sustain proliferation in pre-B-cell acute lymphoblastic leukemia

city: Rome

first: Matteo

last: Pallocca

address: Via Fermo Ognibene

country: Italy

institute: Italian National Cancer Institute "Regina Elena"


title: Che-1 is targeted by c-Myc to sustain proliferation in pre-B-cell acute lymphoblastic leukemia [RNA-seq]

summary: This Series contain trascriptional profiling via RNA-seq for two ALL-B cell lines, one commercial (NALM-6), one patient-derived (LAL-B), in presence or absence of AATF transcript. In NALM-6, also MYC was depleted and sequenced in triplicate. Furthermore, the Burkitt's Lymphoma P493 cell line has been sequenced in presence or absence of Tetracycline (MYC On-Off) and in CN vs siAATF.

overall_design:
RNA-seq of B-cell cell lines after knockdown of Che-1/AATF or MYC.

The submitter declares that the raw data for the LAL-B cell line Samples (GSM2459108-GSM2459113) will be made available in dbGaP (https://www.ncbi.nlm.nih.gov/gap) due to patient privacy concerns.

first: Valentina

last: Folgiero

first: Cristina

last: Sorino

first: Frauke

last: Goeman

first: Francesca

last: De Nicola

first: Franco

last: Locatelli

first: Maurizio

last: Fanciulli


title: Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis [scRNA-seq]

city: La Jolla

first: Pandurangan

last: Vijayanand

address: 9420 Athena Circle

institute: La Jolla Institute for Allergy and Immunology

summary: CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, heterogeneity and clonal diversity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile and clonality in humans. The single-cell differential gene expression analysis, revealed a spectrum of known transcripts, including several linked to cytotoxic and co-stimulatory function, and transcripts of unknown cytotoxicity-related function that are expressed at higher levels in the TEMRA subset, which is highly enriched for CD4-CTLs, compared to cells in the central and effector memory subsets (TCM, TEM). Simultaneous T cells antigen receptor (TCR) analysis in single-cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared to TCM and TEM cells and that the majority of CD4-TEMRA were dengue virus (DENV)-specific in subjects with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across subjects, with four distinct clusters identified by the single-cell analysis. Most importantly, we identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and thus could provide insights into the mechanisms that may be utilized to generate durable and effective CD4-CTL immunity.

overall_design: Transcriptome analysis of  purified populations of human CD4 memory cell subsets by  single-cell RNA-sequencing.

first: Veena

last: Patil

first: Ariel

last: Madrigal

first: Benjamin

last: Schmiedel

first: James

last: Clarke

first: Patrick

last: O'Rourke

first: Eva

last: Harris

first: Aruna

middle: D

last: de Silva

first: Bjoern

last: Peters

first: Grégory

last: Seumois

first: Daniela

last: Weiskopf

first: Alessandro

last: Sette


title: RNA-Seq of Benzalkonium Chloride Treated and Untreated Acinetobacter baumannii

first: Bryan

last: Davies

summary: The goal of this RNA-Seq study was to determine Acinetobacter baumannii's transcriptiional response to sub-MIC concentrations of benzalkonium chloride in Acinetobacter baumannii.  This RNA-seq data was then utilized to aide in the determination of the sub-MIC mechanism of action for benzalkonium chloride.

first: Gregory

last: Knauf


title: Che-1 is targeted by c-Myc to sustain proliferation in pre-B-cell acute lymphoblastic leukemia [ChIP-seq]

first: Matteo

last: Pallocca

summary: This Series contains ChIP-seq for the AATF protein, for NALM-6 and patient-derived LAL-B cell line.

overall_design:
anti-Che-1 ChIP-seq in NALM-6 and LAL-B cell line.

The submitter declares that the raw data for the LAL-B cell line Samples (GSM2459095, GSM2459097) will be made available in dbGaP (https://www.ncbi.nlm.nih.gov/gap) due to patient privacy concerns.

first: Valentina

last: Folgiero

first: Cristina

last: Sorino

first: Frauke

last: Goeman

first: Francesca

last: De Nicola


title: Molecular portraits of tumor mutational and micro-environmental sculpting by immune checkpoint blockade therapy

city: Cleveland

first: Vladimir

last: Makarov

state: OH

address: 9500 Euclid Ave

department: LRI

institute: Cleveland Clinic

summary: Immune checkpoint blockade (ICB) has demonstrated significant promise for the treatment of advanced malignancies. Anti-CTLA4 and ant-PD1 therapy can activate the immune system and result in durable control in diseases such as melanoma and non-small cell lung cancer.

overall_design: 109 RNASeq samples (58 On-treatment and 51 Pre-treatment) from 65 patients

first: Nadeem

last: Riaz

first: Jonathan

last: Have

first: Alexis

last: Desrichard

first: Tymothy

last: Chan


title: Herpes Simplex Virus 1 dramatically alters loading and positioning of RNA Polymerase II on host genes early in infection

status: Public on Jan 22 2018

city: Ithaca

first: Joel

last: Baines

state: NY

address: 235 Hungerfordhill Rd

institute: Cornell University

summary: We used precision nuclear run on (PRO-seq) to determine the location of Pol II at 3 hours post infection with HSV-1 in human epithelial cells.  We found HSV-1 decreased Pol II on approxomately 2/3 of cellular genes, but increased Pol II on others.  For more than 85% of genes for which transcriptional terminatin could be statistically assed, Pol II was displaced to positions downstream of the normal termination zone suggesting extensive termination defects.  Pol II amounts at the promoter, prooter proximal apuse site, and gene bnody were also modulated in a gene-specific manner.

sample_organism:
Drosophila melanogaster
Homo sapiens

platform_organism:
Drosophila melanogaster
Homo sapiens

overall_design: PRO-seq data from two separate experiments looking at Pol II location at 3 hours post infection with HSV-1.  Each experiment had 3 biological replicates of Mock and HSV-1 infected sequencing libraries prepared from Hep-2 cells.  One experiment included a Drosophila nuclei spike-in as a control.

first: Claire

last: Birkenheuer

first: Charles

middle: G

last: Danko


title: Genome-wide identification of starvation responsive genes controlled by SnRK1s and group S1 bZIPs

status: Public on Jan 19 2018

city: Wuerzburg

first: Wolfgang

last: Droege-Laser

address: Julius-von-Sachs Platz 2

department: Julius-von-Sachs Institute

summary: 3 weeks old aseptically grown WT and loss-of-function lines of SnRK1s (transgenic SnRK1α1 T-DNA insertion mutant line crossed with an estradiol inducible amiRNA construct targeting SnRK1α2) and group S1 bZIPs (bZIP1/bZIP53 T-DNA insertion mutant line crossed with an estradiol inducible amiRNA construct simultaneously targeting bZIP2, bZIP11 and bZIP44) were cultivated for 6h under extended night. Total RNA was extracted from whole seedlings and used for RNAseq library preparation.

overall_design: Examination of global transcriptional changes in WT as well as SnRK1 and S1-bZIP knockdown lines in response to short-term dark cultivation.

first: Lorenzo

last: Pedrotti


title: Id proteins suppress E2A-driven iNKT cell development prior to TCR selection [RNA-seq]

first: Yuan

last: Zhuang

summary: Id proteins have been shown to promote the differentiation of conventional αβ and γδT cells, and to suppress the expansion of invariant Natural Killer T (iNKT) cells and innate-like γδNKT within their respective cell lineages. However, it remains to be determined whether Id proteins regulate lineage specification in developing T cells that give rise to these distinct cell fates. Here we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical for the iNKT cell lineage prior to TCR expression. Lack of Id proteins also promotes a biased TCR rearrangement in favor of iNKT cell fate prior to selection at the CD4+CD8+ double positive (DP) stage. Enhanced iNKT development in Id3-deficient mice lacking γδNKT cells suggests that Id3 regulates the lineage competition between these populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT and γδNKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a large population of iNKT cells and a unique innate-like DP population, despite the block in conventional αβ T cell development. The transcriptional profile of these unique DP cells reflects enrichment of innate-like signature genes, including PLZF (Zbtb16) and Granzyme A (Gzma). Results from these genetic models and genome-wide analyses suggest that Id proteins suppress E2A-driven innate-like T cell programs prior to TCR selection to enforce predominance of conventional T cells.

overall_design:
The RNA-Seq experiment included duplicates of WT DP, WT NKT, WT gamma delta, L-DKO DP, L-DKO NKT and Id3KO gamma delta T cells (where L-DKO refers to mice deficient in both Id2/Id3 and Id3KO to mice that lack Id3). Each replicate consisted of pooled cells from 2-3 mice. One WT NKT was removed from analysis due to low quality of sequencing.
The mouse strains for these experiments were either C57BL/6, or C57BL/6 and 129 hybrids.

first: Sumedha

last: Roy

first: Amanda

last: J. Moore

first: Cassandra

last: Love

first: Anupama

last: Reddy

first: Deepthi

last: Rajagopalan

first: Sandeep

last: Dave

first: Leping

last: Li

first: Cornelis

last: Murre


title: RNA sequencing of sorted cells from mouse PDAC

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

institute: CNIO

summary: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplatic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα+ CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts (NPFs) to identify genes potentially implicated in their pro-tumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the acute-phase Serum Amyloid A (SAA) apolipoprotein family, is a key mediator of the pro-tumorigenic activity of PDGFRα+ CAFs. Whereas Saa3 competent CAFs stimulate the growth of PDAC tumor cells in an orthotopic model, Saa3 null CAFs inhibit tumor growth. Saa3 also plays a role in the cross-talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3 null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC tumor development in mice. The pro-tumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases. Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the orthologue of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients.

overall_design: The following groups were used: Normal Pancreatic Fibroblasts (NPFs), Cancer Associated Fibroblasts (CAFs), Saa3 KO CAFs, Saa3 WT tumor cells, Saa3 KO tumor cells. For each group at least 3 biological replicate was included in the analysis.

first: Magdolna

last: Djurec

first: Kevin

last: Troulé

first: Albert

last: Lee


title: Keap1 knockdown in melanocytes induces cell proliferation and survival via HO-1-associated β-catenin signaling

status: Public on Jan 20 2018

city: Seoul

first: Sang Ho

last: Oh

address: 50-1 Yonsei-ro

department: Dermatology

institute: Yonsei University College of Medicine

summary: Nrf2-Keap1 signaling pathway protects cells against photo-oxidative stress. Yet in recent works, its role in melanogenesis together with cell protection functions against oxidative stress has been gaining interest. However, its effect on melanogenesis still has contradictory results from different studies.  The aims of our study were to investigate the effect of Keap1 silencing in melanocyte on melanogenesis and its associated mechanism.  Primary human epidermal melanocytes and melan-a cell line were used for this experiment. RNA sequencing was done to identify genes involved in melanocyte biology using Keap1 knockdown through siRNA techniques. And melanogenesis and the expression of melanogenesis-associated molecules were evaluated in Keap1 silenced melanocyte to examine the effects of Keap1 on melanogenesis, melanocyte growth, and related pathways.  RNA-sequencing data revealed that Keap1 knockdown in primary human epidermal melanocytes (PHEMs) induced cell survival-related gene expression. Additionally, siRNA-mediated inhibition of Keap1 led to upregulation of MITF and melanogenesis-associated molecules along with Nrf2 activation in PHEMs. HO-1, a major gene that is upregulated in RNA-sequencing using Keap1-silenced PHEMs, protected melanocytes against H2O2-induced cell death and upregulated MITF and β-catenin expression. Further, increased expression of melanogenesis-associated molecules after Keap1 silencing was validated to occur through HO-1-associated β-catenin activation in a Keap1 and HO-1 double knockdown experiment.  This work suggests that Keap1 silencing in melanocytes induced melanogenesis and the expression of melanogenesis-associated molecules through HO-1-associated β-catenin activation. Keap1 downregulation in melanocytes is important for cell proliferation and survival.

overall_design: Total mRNAs were directly extracted from primary human melanocytes with or without silencing of Keap1 expression. Isolated RNA was labelled and used for RNA-sequencing.

first: Sang

middle: H


title: Id proteins suppress E2A-driven iNKT cell development prior to TCR selection [ChIP-seq]

overall_design:
The ChIP-Seq experiment included E2A ChIP-Seq of DP and iNKT cells from L-DKO mice (deficient in Id2 and Id3). Each sample consisted of pooled cells from 3-4 mice. Input controls (i.e. not immunoprecipitated with E2A antibody) were included for each sample.
The mouse strains for these experiments were either C57BL/6, or C57BL/6 and 129 hybrids.

last: Moore


title: Transcriptional response of C.trachomatis to early-cycle iron-starvation

city: PULLMAN

middle: Jo

last: Brinkworth

state: WA

address: 1770 NE Stadium Way

department: School of Molecular Biosciences

institute: Washington State University

summary: Early-cycle C. trachomatis differentially regulates a subset of genes in response to iron-starvation

overall_design: C.trachomatis infected cells were mock-treated or treated with 100 micromolar BPDL for 3h, starting at 6h post-infection

first: Mark

last: Wildung


title: Transcriptional response of C.trachomatis to mid-cycle iron-starvation

summary: Mid-cycle C. trachomatis differentially regulates a subset of genes in response to iron-starvation

overall_design: C.trachomatis infected cells were mock-treated or treated with 100 micromolar BPDL for 3 or 6h, starting at 12h post-infection


title: KSHV chromatin looping facilitates effective gene expression: high-resolution mapping of K-Rta binding sites in the KSHV genome [ChIP-seq]

city: Sacramento

first: Clifford

middle: G.

last: Tepper

state: CA

address: 4645 2nd Avenue, Room 2300A

department: Biochemistry and Molecular Medicine

institute: UC Davis School of Medicine

summary: The three-dimensional structure of chromatin via formation of genomic loops allows cellular RNA polymerase II to access distal promoters, thus it has a significant impact on the outcome of gene expression. The Kaposi’s sarcoma-associated herpesvirus (KSHV) entire lytic transcriptional program of 80 plus genes is initiated by a single viral transactivator, K-Rta. Here we examined the relationship between the KSHV genomic structure and K-Rta recruitment sites with unbiased Capture Hi-C analyses. The studies showed that KSHV forms a unique genomic structure, which coordinates a domain-specific viral gene expression program via K-Rta recruitment, and identified a number of direct physical, long-range, and dynamic genomic interactions. Chromatin immunoprecipitation-sequencing analyses identified a limited number of K-Rta recruitment sites in the KSHV genome, including the K12 and PAN RNA promoters. KSHV engineered to harbor point mutations in the K-Rta-responsive elements (RE) at these promoters significantly attenuated not only the direct downstream gene, but also distal viral gene expression in a domain-specific manner. Despite the long distance between two RE sites in the linear orientation, efficient recruitment of K-Rta to either promoter required intact K-Rta REs at both promoters. Finally, inducible genomic loops generated during KSHV reactivation occurred preferentially at K-Rta recruitment sites. Mutation of a K-Rta RE inhibited formation of inducible genomic loops, gene expression of its destination of looping genes, and KSHV replication in de novo infected human gingival epithelial cells. These results suggest that the KSHV genome is programed to form both stable and inducible chromatin hubs for effective KSHV gene expression, and partly explains why K-Rta has such a dominant effect on KSHV lytic gene transcription.

overall_design: The goal of these studies was to perform genome-wide mapping of K-Rta binding sites across the KSHV genome using ChIP-Sequencing (ChIP-Seq). This was performed in the context of the BCBL-1 cell line model, which is derived from a KSHV-infected human primary effusion lymphoma (PEL) and contains latent KSHV genomes. For these studies, we utilized an engineered subline, referred to as TREx-K-Rta BCBL-1, which contains a Tetracycline/Doxycycline (Dox)-inducible Flagx3- and HAx3-tagged K-Rta expression cassette. K-Rta expression was induced in individual TREx-K-Rta BCBL-1 cultures by treatment with Dox for 24 hours. Following treatment, the cells were then processed for ChIP-Seq analyses by formaldehyde cross-linking, chromatin solubilization, and chromatin immunoprecipitation (ChIP) with anti-FLAG M2 monoclonal antibody. Subsequently, DNA was isolated from the ChIP and total chromatin (Input) samples, and followed by library preparation and next-generation sequencing (NGS).

first: Yoshihiro

last: Izumiya

first: Mel

last: Campbell


title: RNA G-quadruplex secondary structure promotes alternative splicing via the RNA binding protein hnRNPF

first: Samuel

middle: Emerson

last: Harvey

state: TEXAS

department: Lester and Sue Smith Breast Center

summary: It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex forming capacity promote exon inclusion. Destroying G-quadruplex forming capacity while keeping G-tracts intact abrogates exon inclusion. Analysis of RNA binding protein footprints revealed that G-quadruplexes are enriched in hnRNPF-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an EMT-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA datasets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G-quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression.

overall_design: PolyA-RNA-sequencing of two non-specific shRNA and two shhnRNPF HMLE cell lines

first: C

last: Cheng

first: S


title: Transcriptional response of C.trachomatis to early-cycle and mid-cycle iron-starvation

first: Amanda

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

overall_design: Refer to individual Series


title: Aberrant mRNA m6A methylation promotes the progression of endometrial cancer by altering Akt activity

city: Chicago

first: zhike

last: lu

state: IL

address: 5801 South Ellis Avenue

department: department of chemistry

institute: University of Chicago

summary: The N6-methyladenosine (m6A) methylation of mRNA is an emerging gene regulatory mechanism that controls the stability and translation of methylated transcripts in development and cell differentiation. Here, we show that aberrant m6A methylation plays key roles in the progression of endometrial cancer. About 70% of endometrial tumors exhibit reduced m6A methylation due to either mutation in METTL14 (~10%) or decreased expression of METTL3 (~60%). The reduction of mRNA m6A methylation through METTL14 mutation or METTL3 downregulation increased the proliferation and tumorigenicity of endometrial cancer cells in vitro and in vivo. RNA-seq and m6A-seq of human tumor tissue identified several transcripts in the Akt pathway exhibiting altered m6A methylation, including PHLPP2 and components of mTORC2. The altered stability and translation of these transcripts mediated the increased proliferation of cells with reduced mRNA m6A methylation. These results establish aberrant m6A methylation as a critical oncogenic mechanism underlying a majority of endometrial cancer.

overall_design: m6A in endometrial cancer patient samples and HEC-1-A stable cell lines

first: Jun

last: Liu

first: Bryan

middle: T

last: Harada

first: Zhike

last: Lu

first: Chuan

last: He


title: TCF7L1 ChIP-seq in H9 human embryonic stem cells

city: Irvine

first: Peter

last: Donovan

address: Gross Hall, Rm 3006, 845 Health Sciences Road

institute: University of California, Irvine

summary: Genome-wide binding profile of TCF7L1 in human embrynoic stem cells.

overall_design: Chip-seq was performed on endogenous TCF7L1 in H9 human embryonic stem cells.  Two biological replicates of the TCF7L1 ChIP-seq were performed and one Input (no immunoprecipitation) biological replicate was performed.

first: Robbie

last: Sierra

first: Nate

last: Hoverter


title: Assessment of Su(H) binding sites in the Drosophila S2-DRSC cell line

city: Basel

first: Renato

last: Paro

address: Mattenstrasse 35

department: Department of Biosystems Science and Engineering (D-BSSE)

country: Switzerland

institute: Swiss Federal Institute of Technology Zurich (ETHZ)

summary: We performed ChIP-sequencing with antibodies targeting Su(H) to assess the genomic binding sites of this protein in S2 cells as a reference for the comparison with published data of this cell line

overall_design: Chromatin from S2 cells was immunoprecipitated using antibodies against Su(H)

first: Alexander

middle: B

last: Schwahn


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild type and RBM3 knockout cortex Transcriptomes during maternal hypothermia

city: Beijing

first: Jianwei

last: Jiao

state: Beijing

address: 1 Beichen West Road

department: State Key Laboratory of Reproductive Biology

institute: Institute of Zoology, Chinese Academy of Sciences

summary: Next-generation sequencing (NGS) has revolutionized systems-based analysis of gene expression. The goals of this study are to compare the different transcripts between Wild type and RBM3 knockout cortex Transcriptomes during maternal hypothermia

overall_design: Methods:the maternal hypothermia was induced at E14, and the embryonic brain cortex were isolated from E16 WT and RBM3 knockout mice. RNA used for global transcriptome analysis were extracted from the embryonic brain cortex and sequenced by Hiseq 2500. Significantly differentially expressed genes were identified when we compared Normalized Reads Count between groups with p < 0.05 and |Log2FoldChange| > 1.

first: Wenlong

last: Xia

first: Libo

last: Su


title: A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets.

first: Ángel

middle: F

last: Álvarez-Prado

address: Melchor Fernández Almagro, 3

institute: Centro Nacional de Investigaciones Cardiovasculares

summary: Activation Induced Deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in germinal center (GC) B cells through the deamination of deoxycytidine residues (dC) into deoxyuridines (dU) in immunoglobulin (Ig) genes. Although AID has a strong preference for Ig genes, it can also target other genomic regions, giving rise to mutations or chromosomal translocations. Thus, understanding the specificity of AID has major implications for oncogenic transformation. However, approaching AID specificity has proved extremely challenging because AID deamination events occur at low frequencies. Here we have sequenced at very high depth >1500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. This has enabled for the first time to define the molecular features predictive of AID target specificity genome-wide. Furthermore, we identify the most highly mutated hotspot for AID activity described to date. We also find that Base Excision Repair (BER) and Mismatch Repair (MMR) systems, which are responsible for the resolution of AID deaminations, back-up each other to faithfully repair AID-induced lesions. Finally, our data establishes a novel link between AID mutagenic activity and malignant transformation.

overall_design: Germinal center (CD19+FAS+GL7+) B cells were sorted from Peyer's patches of Ung+/-Msh2+/-, Ung+/-Msh2-/-, Ung-/-Msh2+/-, Ung-/-Msh2-/- and Aicda-/- mice. Genomic DNA was isolated, enriched with a custom SureSelect library of ~1600 RNA probes and deep sequenced. Four groups of mice were analzyed in 2 replicates; Aicda-/- mice were used as negative controls.

first: A

last: Ramiro


title: Environmental enrichment increases transcriptional and epigenetic differentiation between mouse dorsal and ventral dentate gyrus neurons

city: San Diego

first: Eran

middle: A

last: Mukamel

state: United States

address: 9500 Gilman Dr.

department: Cognitive Science

institute: University of California-San Diego

summary: We examined molecular genetic differences between the dorsal and ventral dentate gyrus of the hippocampus in mouse, using RNA-seq to assess differences in gene expression, as well as MethylC-seq and TAB-seq to assess differences in DNA methylation and hydroxymethylation, respectively. In addition, we examined how an enriched environment can engender changes in gene expression and methylation.

overall_design: RNA-seq, MethylC-seq, TAB-seq in dorsal and ventral dentate gyrus of the hippocampus

first: Tie-Yuan

last: Zhang

first: Christopher

middle: L

last: Keown

first: Junhao

last: Li

first: Xianglan

last: Wen

first: Dulcie

last: Vousden

last: Anacker

first: Richard

last: Ryan

first: Josie

last: Diorio

first: Nicholas

last: O'Toole

first: Jason

last: Lerch

first: Michael

middle: J

last: Meaney


title: Changes at the nuclear lamina alter binding of pioneer factor Foxa2 in aged liver.

city: Charlottesville

first: Irina

last: Bochkis

address: 1340 Jefferson Park Ave

department: Pharmacology

institute: University of Virginia

summary: Increasing evidence suggests that regulation of heterochromatin at the nuclear envelope underlies metabolic disease susceptibility and age-dependent metabolic changes, but the mechanism is unknown. Here we profile lamina-associated domains (LADs) using lamin B1 ChIP-Seq in young and old hepatocytes and find that, although lamin B1 resides at a large fraction of domains at both ages, a third of lamin B1-associated regions are bound exclusively at each age in vivo. Regions occupied by lamin B1 solely in young livers are enriched for the forkhead motif, bound by Foxa pioneer factors. We also show that Foxa2 binds more sites in Zmpste24 mutant mice, a progeroid laminopathy model, similar to increased Foxa2 occupancy in old livers. Aged and Zmpste24-deficient livers share several features, including nuclear lamina abnormalities, increased Foxa2 binding, de-repression of PPAR and LXR-dependent gene expression, and fatty liver. In old livers, additional Foxa2 binding is correlated to loss of lamin B1 and heterochromatin (H3K9me3 occupancy) at these loci. Our observations suggest that changes at the nuclear lamina are linked to altered Foxa2 binding, enabling opening of chromatin and de-repression of genes encoding lipid synthesis and storage targets that contribute to etiology of hepatic steatosis.

overall_design: Genome-wide location analysis (ChIP-Seq) of lamin B1, H3K9me3, and H3K9me2 from young (3 months) and old (21 months) mouse livers and Foxa2 in wildtype and Zmpste24 KO  12-week mouse livers .

first: Larry

last: Singh


title: Evidence for rRNA 2'-O-methylation plasticity: control of intrinsic translational capabilities of human ribosomes

city: ORSAY

first: Pierre

last: Bertin

address: Batiment 400, Université Paris Sud

country: France

institute: Institut de Biologie Intégrative de la Cellule

summary: Ribosomal RNAs (rRNAs) are main effectors of mRNA decoding, peptide-bond formation and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and display a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin (FBL) in human cells. Using RiboMethSeq, a non-biased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus (CrPV) IRES element, as a model, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through 2'-O-Me pattern and not by non-ribosomal actors of the translational machinery. Our results establish rRNA 2'-O-methylation plasticity as a mechanism providing functional specificity to human ribosomes.

overall_design:
RNAseq was conducted to get the landscape of transcripts abundances.
RIBOseq was conducted to observe the ribosome occupancy on each protein-coding gene of the hg38 genome assembly.

first: Jenny

last: Erales

first: Virginie

last: Marchand

first: Baptiste

last: Panthu

first: Sandra

last: Gillot

first: Stéphane

last: Belin

middle: E

last: Ghayad

first: Maxime

last: Garcia

first: Florian

last: Laforêts

last: Marcel

first: Agnès

last: Baudin-Baillieu

first: Yohann

last: Coute

first: Annie

last: Adrait

first: Mélanie

last: Meyer

first: Gabriel

last: Therizols

first: Marat

last: Yusupov

first: Olivier

last: Namy

first: Théophile

last: Ohlmann

first: Yuri

last: Motorin

first: Frédéric

last: Catez

first: Jean

last: Diaz


title: Loss of Capicua alters early T cell development and predisposes mice to T cell lymphoblastic leukemia/lymphoma

status: Public on Jan 18 2018

city: Edmonton

first: Qiumin

last: Tan

state: Alberta

address: 6-16 Medical Sciences Building

department: Cell Biology

country: Canada

institute: University of Alberta

summary: Thymic lymphomas were dissected from Cic adult knockout mice or wildtype mice transplanted with Cic adult knockout progenitors. Tissues were snap-frozen in liquid nitrogen until further analysis. RNAs were purified using the AllPrep DNA/RNA mini kit (Qiagen).

overall_design: Examination of gene expression in Cic-null lymphomas versus normal thymus from control littermates

first: Ying-Wooi

last: Wan

first: Huda

middle: Y

last: Zoghbi


title: Base-resolution analysis of DNA methylation patterns downstream of Dnmt3a in mouse naïve B cells

city: Research Triangle Park

first: Paul

last: Wade

address: 111 TW Alexander Drive

department: Laboratory of Molecular Carcinogenesis

institute: NIEHS

summary:
Background: The DNA methyltransferase, Dnmt3a, is one of the most commonly mutated genes in adult hematologic malignancies and is dynamically regulated throughout normal B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. While a cell-type specific role for Dnmt3a has been proposed in carcinogenesis, the role of Dnmt3a in directing specific DNA methylation patterns in B cell development has not been characterized.
Results: We selectively inactivated Dnmt3a early in B cell development and then utilized Whole Genome Bisulfite Sequencing to generate base-resolution profiles of Dnmt3a+/+ and Dnmt3a−/−  mouse naïve splenic B cells. Although overall methylation level distribution was largely consistent between Dnmt3a+/+ and Dnmt3a−/− B cells, we identified site-specific and regional differences corresponding to both gains and losses of methylation in Dnmt3a−/− cells relative to Dnmt3a+/+ cells. Hypomethylated regions (N = 313) in Dnmt3a−/− cells, however, were more frequently observed than were hypermethylated regions (N = 136) by over 2-fold, consistent with loss of methyltransferase activity.
Conclusions: We have identified a set of CpG sites that require Dnmt3a to maintain normal methylation levels in naïve B cells.  These CpGs are associated with genes expressed in B cells that undergo dysregulation in leukemias.

overall_design: Base-resolution methylomes for splenic naïve B cells from adult Dnmt3a+/+ and Dnmt3a−/− mice using Whole Genome Bisulfite Sequencing


title: miR-204-5p and miR-211-5p contribute to BRAF inhibitor resistance in melanoma

first: Marta

last: Diaz Martinez

state: Madrid

address: Ramiro de Maeztu, 9

institute: Centro de Investigaciones Biológicas

summary:
Purpose:  Identification of miRNAs that enable resistance to BRAF inhibitors in melanoma suggests a mechanism-based strategy to limit resistance and improve clinical outcomes.
Methods: We generated A375 melanoma cells resistant to Vemurafenib (VMF) with the goal of investigating changes in miRNA expression patterns that might contribute to resistance.
Results: Increased expression of miR-204-5p and miR-211-5p occurring in VMF-resistant cells was determined to impact VMF response.

overall_design: miRNA profiles of parental and VMF-resistant melanoma cell lines were generated by deep sequencing, in duplicate, using Illumina HiSeq.

last: Díaz-Martínez

first: Lucía

last: Benito-Jardón

first: Lola

last: Alonso

first: Lisa

last: Koetz-Ploch

first: Eva

last: Hernando

first: Joaquin

last: Teixidó


title: Enhanced pre-clinical anti-tumor activity of M7824, a novel bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β

city: Billerica

first: Chunxiao

last: Xu

state: MA

address: 45A Middlesex Tpke

institute: EMD Serono Research and Development Institute

summary: EMT-6 orthotopic tumors were grown in Balb/C mice.  Mice were randomized into one of four treatment arms (treatment on days 0,1,2).  Tumors were collected on day 6.

overall_design: 8-12 week old Balb/C females were inoculated with 0.25x10^6 viable EMT6 cells in 0.1 mL PBS into the right mammary pad.  Dosing was initiated on day 0 (20 days after tumor cell inoculation), when the mean tumor volume reached ~400 mm^3.

first: Yan

last: Lan

first: Bo

last: Marelli

first: Jin

last: Qi

first: Huakui

last: Yu

first: Guozhong

last: Qin

first: Alex

last: Rolfe

first: George

last: Locke


title: Defining the physiological role of SRP in protein-targeting efficiency and specificity

city: Cambridge

first: Jonathan

middle: S

last: Weissman

address: 455 Main St

department: HHMI, Biology

institute: Whitehead Institute for Biomedical Research, MIT

summary: The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP’s in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino-terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP’s absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP’s essential roles in maintaining the efficiency and specificity of protein targeting.

overall_design: Ribosome profiling of whole cell or streptavidin-purified ribosomes biotinylated by ER or mitochondrial localized biotin ligase in yeast

first: Elizabeth

last: Costa

first: Kelly

last: Subramanian

first: Jodi

last: Nunnari


title: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes of human U2OS cells under mild replication stress by low dose aphidicolin (APH)

city: Kyoto

first: Masamichi

last: Ishiai

state: Kyoto

address: Yoshidakonoe-cho, Sakyo-ku

department: Radiation Biology Center

country: Japan

institute: Kyoto University

summary: To detect transcripts before and after APH treatment, we subjected total RNA isolated from U2OS cells expressing human FANCD2-3xFLAG to next generation sequencing.

overall_design: U2OS cells expressing human FANCD2-3xFLAG  were treated with 0.4 micro M APH, or left antreated for 24 hrs.

first: Yusuke

last: Okamoto

first: Watal

middle: M

last: Iwasaki

first: Kazuto

last: Kugou

first: Chinatsu

last: Ohki

first: Kunihiro

last: Ohta

first: Hideki

last: Innan

first: Minoru

last: Takata


title: Characterization of miRNomes in two mouse Embryonic Stem Cell lines subjected to differentiation

city: Warsaw

first: Joanna

middle: Maria

last: Bem

address: Miecznikowa 1

department: Department of Cytology

country: Poland

institute: University of Warsaw

summary: We used high throughput miRNA sequencing to investigate differences between two mESC lines (D3 and B8 ESCs). Undifferentiated ESCs, cells differentiating in embryoid bodies (day 7), and also cells from embryoid bodies outgrowth (day 21) were analyzed.

overall_design: ESCs of both cell lines were differentiated in medium containing e.g. retinoic acid, insulin, transferrin, selen and N2 supplement.

first: Anna

last: Fogtman

first: Maria

last: Ciemerych


title: Nr2f transcription factors promote the identity  of the vertebrate upper jaw

city: Los Angeles

first: Gage

middle: DeKoeyer

last: Crump

address: 1425 San Pablo St BCC403

department: Stem Cell Biology and Regenerative Medicine

institute: University of Southern California

summary: The jaws are complementary in form and function but develop asymmetrically, as the lower but not upper jaw bone forms around a prominent cartilage template. How such differences in skeletal composition are patterned is unclear. Here, we identify four Nuclear receptor 2f genes, nr2f1a, nr2f1b, nr2f2, and nr2f5, as enriched in zebrafish upper jaw precursors. Whereas loss of Nr2f genes results in expansion of upper jaw cartilage to resemble that of the lower jaw, Nr2f5 misexpression inhibits lower jaw cartilage formation. Genome-wide analyses show that Nr2f genes prevent expansion of lower jaw-associated gene expression into the upper jaw territory. Further, restriction of Nr2f expression by Endothelin1 signaling is critical for lower jaw development, as reducing Nr2f dosage fully restores lower jaw development in edn1 mutants. We propose that Nr2f genes drive jaw asymmetry by limiting early cartilage differentiation in the upper jaw to preserve more precursors for later osteogenesis.

overall_design: Zebrafish pharyngeal arch cells expressing both fli1a:GFP and sox10:DsRed were isolated by FACS from 36 hpf nr2f2; nr2f5 double mutant embryos (2 replicates: n = 37 & 84; 18,858 & 30,655 cells), sox10:Gal4+ embryos (n = 69; 86,517 cells), sox10:Gal4; UAS:Nr2f5 embryos (n = 84; 56,866 cells).

first: Lindsey

last: Barske

first: Pauline

last: Rataud

first: Samuel

last: Cox

first: Kasra

last: Behizad

first: J


title: Telomerase-expressing distributed hepatocyte stem cells repopulate the liver during homeostasis and injury

city: Stanford

first: Shengda

last: Lin

address: 269 Campus Dr. CCSR 1150

department: Medicine

institute: Stanford University

summary: We identified a subset of hepatocytes with high Telomerase Reverse transcriptase (Tert) that functions as the repopulating stem cells in homeostasis and injury. We performed RNA-Seq to reveal the differences of these cells and the other hepatocytes.

overall_design: RNA mRNA profiles of TERT(High) and TERT (Low) hepatocytes from 2-month old mice were generated by deep sequencing, in triplicate, using Illumina platform.

first: Alina

last: Garbuzov

first: Steven

last: Artandi


title: Nudix-type RNA pyrophosphohydrolase regulates pathogenesis-associated factors in Pseudomonas aeruginosa

first: Maciej

last: Lirski

address: Pawińskiego 5a

department: Department of Protein Biosynthesis

institute: Institute of Biochemistry and Biophysics, Polish Academy of Sciences

summary: The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from E. coli which removes pyrophosphate from 5’-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similarly to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in down-regulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type, but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild type strain were compared using RNA sequencing. The cellular level of a number of transcripts was affected by the lack of PA0336. We focused our attention on pathogenesis-related genes. Up-regulated in the PA0336 mutant were transcripts coding for, i. a., proteins involved in the regulation and/or production of pyocyanin, biofim-associated alginates and exotoxins. The results from the global analysis were verified by determining the cellular level of chosen transcripts by quantitative RT-PCR method. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence factors.

overall_design: Study comparing RNA expression of P. aeruginosa PA0336 mutant strain with wild type reference, both in biological triplicates, by RNA-seq performed on Ion Torrent Proton platform

first: Martyna

last: Kujawa

first: Mateusz

last: Zięcina

last: Drabińska

first: Marta

last: Modzelan

first: Elżbieta

last: Kraszewska


title: Transcriptional profile of mouse nephron progenitor cells

first: Andrew

middle: P

last: McMahon

state: California

address: 1425 San Pablo St, BCC 312

department: Stem Cell Biol & Regen Med

summary: In developing mammalian kidney, nephron progenitor cells (NPC) give rise to all cells in mature nephrons. Expression of the transcription factor Six2 marks NPC in developing mouse kidneys. Within the Six2+ cell population, uncommitted NPC is marked by Cited1 expression. Towards the depletion of NPC in P2, most of the Cited1+ NPC is committed. Therefore, most of the Six2+ cells in kidney represents committed NPC. In order to explore the mechanism of NPC self-renewal and differentiation, hereby we generated transcriptionl profiles of uncommitted NPC (Cited1RFP+ cells from kidneys of E16.5 Cited1tagRFP transgenic mice) and committed NPC (Six2GFP+ cells from kidneys of P2 Six2TGC transgenic mice).

overall_design: To obtain transcriptional profile of the target cells, we performed mRNA-Seq of cells purified from transgenic mouse kidneys (Six2TGC, Cited1tagRFP) using FACS.

first: Qiuyu

last: Guo

first: Albert

last: Kim


title: Mapping of transcription factor binding sites in mouse nephron progenitor cells

summary: In developing mammalian kidney, nephron progenitor cells (NPC) give rise to all cells in mature nephrons. Several transcription factors, including Six2, Hoxd11, Osr1 and Wt1, are expressed in NPC and are essential for its maintenance and/or specification. In order to understand the regulatory functions of these factors, we mapped binding sites of Six2, Hoxd11 and Osr1 in NPC using a novel transgenic strategy, and of Wt1 in wild-type developing kidney.

overall_design: We generated transgenic mice ectopically expressing Six2, hoxd11 or Osr1, driven by an enhancer specifically regulate Six2 expression. In the transgenic mice, each transgenic transcription factor is tag with the FLAG epitope at the 3'-end.  To genome-wide map binding sites of Six2, Hoxd11 and Osr1 specifically in mouse nephron progenitor cells, we performed FLAG ChIP using embryonic kidney isolated from these mice. To map Wt1 binding sites in kidney, we independently performed Wt1 ChIP-Seq in wild-type mouse kidneys.

first: Lori

last: O'Brien

first: Joo-Seop

last: Park


title: Ribosome profiling of HEK293 cells.

city: Houston

first: Yiwen

last: Chen

state: Texas

address: 1400 Pressler St

department: Bioinformatics and computational biology

institute: UT MD Anderson Cancer Center

summary: The development of the ribosome profiling (ribo-seq) technique has enabled the measurement of translation at a genome-wide level. There are several variants of the ribosome profiling technique that use different translation inhibitors. The regular ribo-seq utilizes Cycloheximide (CHX), a translation elongation inhibitor to freeze all translating ribosomes. In contrast to CHX, the translation inhibitor lactimidomycin (LTM) and harringtonine (Harr) have a much stronger effect on initiating ribosomes. The use of these inhibitors allows for the global mapping of translating initiating sites (TISs) when they are coupled with ribosome profiling (TI-seq). We have developed a computational tool to detect and/or quantitatively compare translation initiation from TI-seq data, and predict novel ORFs from regular CHX-based ribo-seq data. Two replicates of CHX-based ribo-seq experiments and one Harr based ribo-seq were performed in HEK293 cells for confirming the ORFs predicted using public available data.

overall_design: Ribosome protected fragments were extracted from HEK293 cells treated with cycloheximide or harringtonine plus cycloheximide.

first: Dandan

last: He

first: Peng

last: Zhang


title: Accelerated and Improved Differentiation of Retinal Organoids from Mouse Pluripotent Stem Cells in Rotating-Wall Bioreactors

city: Bethesda

first: Matthew

last: Brooks

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

summary: Pluripotent stem cells can be differentiated into three-dimensional (3D) retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall bioreactors (RWB) to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWB demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWB organoids at day (D)25 reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

overall_design: RNA-seq characterization of embryonic stem cell derived retina organoids cultured from intact static suspension culture (SSCi) from day(D) 18 through D32, dissected static suspension culture (SSCd) from D18 through D28, and from rotating-wall bioreactor (RWB) from D18 through D25. Experiments were performed either twice or three times and considered biological replicates. Total RNA was purified from tissue homogenates  using TriPure isolation reagent (Roche, Basel, Switzerland). RNA quantity was assessed with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Rockville, MD). Libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego CA) and paired-end sequenced at 125 bases on a HiSeq 2500.

first: Tyler

last: Di Stefano

first: Holly

first: Christopher

last: Panebianco

first: Koray

middle: D

last: Kaya

first: Linn

last: Gieser

first: Nicole

last: Morgan

first: Tom

last: Pohida

first: Anand

last: Swaroop


title: The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes

status: Public on Jan 17 2018

city: London

first: Helen

last: Rowe

address: 90 Gower Street

department: Infection and Immunity

institute: University College London

summary: Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of a SVA retrotransposon reporter. By using naïve vs. primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. While the HUSH component TASOR binds to endogenous retroviruses (ERVs) and L1s, it is mainly required to repress evolutionarily young L1s. TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and TASOR are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon non-coding DNA.

overall_design: Dissection of which transposons and genes the HUSH complex and TRIM28 co-regulate

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