title: Autonomous TNF is critical for monocyte survival in steady state and inflammation in vivo

city: REHOVOT

first: Steffen

last: Jung

address: Herzl

department: Immunology and Regenerative Biology

country: Israel

institute: The Weizmann Institute of Science

summary: Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are upregulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such the spinal cord during experimental autoimmune encephalomyelitis (EAE).  Using competitive in vitro and in vivo assays, we show here that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes / macrophages, but not in microglia delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6Chi effector monocytes.  Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance and function.

overall_design: BM precursors and steady state monocytes are taken and compared with effector spinal cord monocytes in EAE day 9

first: Yochai

last: Wolf

first: Alexander

last: Mildner


title: Catch-and-release of cytokines converts transient exposure into long-term inflammatory responses

status: Public on Apr 18 2017

city: Bethesda

first: Jennifer

last: Oyler-Yaniv

state: MD

address: 37 Convent Drive, Bldg. 37, Room 4137

department: National Cancer Institute

country: USA

institute: National Institutes of Health

type: Expression profiling by high throughput sequencing

summary: We report that after cancer cells are exposed to a transient pulse (5h) of the pro-inflammatory cytokine IFNγ, they continue up-regulating genes in the antigen presentation pathway for days after abrogation of the original signal. We discovered that a new mechanism of cell-to-cell communication, cytokine catch-and-release, explains this surprising result.

overall_design: Examination of IFNγ-induced gene expression over multiple timepoints in mouse melanoma cells

first: Grégoire

last: Altan-Bonnet


title: Polycomb repressive complex 1 (PRC1) mediated regualtion of uterine stromal cells decidualization.

city: Cincinnati

first: Artem

last: Barski

state: OH

address: 3333 Burnet Av

department: Allergy

institute: Cincinnati Children's Hospital

summary: We extract RNA from mouse implantation sites (IS) on D8 of pregnancy. Mice were treated with or without PRT4165, an inhibitior of PRC1. Total RNAs were analyzed by RNA sequncing.

overall_design: There are four samples to be analyzed:Control group 1) "D8ISCOT-1" and 2) "D8ISCOT-2” ; PRT4165 treated groups 3) "D8ISPRT4165-1", 4)”D8ISPRT4165-2”

first: Fenghua

last: Bian

first: Fei

last: Gao

first: Andrey

last: Kartashov

first: Sanjoy

last: Das


title: Gastric cancer RNA-seq

city: ChiaYi

first: Chin

last: Li

address: 168 University Road, Minhsiung

department: Life Science

country: Taiwan

institute: National Chung Cheng University

summary: RNA  sequencong of 9 upper GI gastric biopsies and resected gastric tissues

overall_design: Total RNA was extracted from clinical specimens. Samples with RIN above 7 were included for library construction using Agilent SureSelect Strand Specific RNA Library Preparation Kit.

first: Yung-Yu

last: Hsieh

first: Shui-Yi

last: Tung

first: Yi-Fang

last: Deng

first: Wan-Ting

last: Hsu


title: Gastric cancer targeted RNA-seq

summary: targeted gene sequencong of 157 preselected genes in upper GI biopsies and resected stomack tissues

overall_design: Total RNA was extracted from clinical specimens. Samples with RIN above 3 were included for library construction using Illumina Truseq targeted RNA expression kits.


title: RNA-seq analysis revealed aberrant gene expression in motor neurons derived from ALS patient iPSCs bearing SOD1+/A272C mutation

status: Public on Apr 17 2017

city: Beijing

first: wang

middle: li

last: xia

address: 15 Datun Road, Chaoyang District

department: Institute of Biophysics

country: China

institute: University of Chinese Academy of Sciences

summary: The goal of this study is to gain insight into the early biomarkers and molecular pathways affected by the SOD1+/A272C mutation in human motor neurons. Isogenic control line was created by CRISPR/Cas9 mediated targeted gene correction. Motor neurons were derived from isogenic iPSC lines, and RNA sequencing was employed to determine differentially expressed genes. This study provides an isogenic platform to study ALS disease mechanism at the early stage.

overall_design: wild type and mutant, 2 replicates.

first: Lixia

last: Wang

first: Guang-Hui

last: Liu


title: Small RNA Sibling ryfA1 Influences Shigella dysenteriae Pathogenesis

city: Athens

first: Ronan

middle: Kevin

last: Carroll

state: OHIO

address: 107 Irvine Hall

department: Biological Sciences

institute: Ohio University

summary: Small regulatory RNAs (sRNAs) of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the over production of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread and the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1; the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.

overall_design: Two RNAseq data sets in total.  Each sample was generated by pooling three independent biological replicate RNA preps

first: Megan

middle: E

last: Fris

first: William

middle: H

last: Broach

first: Sarah

last: Klim

first: Peter

middle: W

last: Coschigano

middle: K

first: Clayton

middle: C

last: Caswell

first: Erin

middle: R

last: Murphy


title: Differential expression of microRNAs in wildtype versus DCL1 mutants in Arabidopsis thaliana

city: Oxford

first: Irina

last: Mohorianu

address: Headington

department: Medical Sciences Division

country: United Kingdom

institute: University of Oxford

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNA libraries of wildtype Arabidopsis thaliana and its mutant Dicer-like 1 (Dcl1) were constructed and sequenced for miRNA identification and expression analysis. The mutant data was used to validate novel miRNA predictions (from miRCat2 (Paicu et al. 2017), miRCat (Moxon et al. 2008), miRPlant (An et al. 2014) and miReap (http://mireap.sourceforge.net/)), by calculating the log fold change between the mutant and the wildtype samples.

overall_design: Seeds of Arabidopsis thaliana wildtype and dcl1-7 mutants were stratified and germinated. When plants were grown, leaves were harvested and genomic DNA were extracted as previously described (Amani et al. 2011) for genotyping. Heterozygous individuals were identified through PCR-sequencing for seed increase (mutants) and by phenotypes (wildtype). When the flower buds in both wildtype and mutant plants were visible but without opening flowers, the leaves of nine plants were harvested for three biological replicates. One replicate consists of a pool of leaves from three individual plants. Total RNA of both wildtype and mutant leaf tissue was extracted, then small RNA (sRNA) of each sample was further enriched and used for HD library construction. The small RNA libraries were sequenced and analyzed.

first: Claudia

last: Paicu

first: Matthew

last: Stocks

first: Ping

last: Xu

first: Aurore

last: Coince

first: Martina

last: Billmeier

first: Tamas

last: Dalmay

first: Vincent

last: Moulton

first: Simon

last: Moxon


title: Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

city: Burlington

first: Jonathan

middle: AR

last: Gordon

state: VT

address: 89 Beaumont Ave Given E209

department: Biochemistry

institute: University of Vermont

summary: The Runx1 transcription factor is essential for hematopoietic differentiation and mutations underlie various leukemias. Here we demonstrate a role for Runx1 in the MCF10 cell series model of breast cancer progression. The highest level of Runx1 that occurs in normal like mammary epithelial cells (MCF10A) is decreased in tumorigenic (MCF10AT1) and metastatic (MCF10CA1a) breast cancer cells. We show that depletion of Runx1 in MCF10A cells results in striking changes in cell morphology and induction of epithelial-mesenchymal transition (EMT) via several signaling pathways. Analyses of breast tumors and patient survival data reveal that loss of Runx1 is associated with poor prognosis and decreased survival. Re-expressing Runx1 in MCF10AT1 breast cancer cells restores the epithelial phenotype. These results identify a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT. These mechanisms suggest Runx1 levels in early stage tumors can be used as a prognostic indicator of tumor progression.

overall_design: RNA-Seq; two replicates each sample (6 total)

first: Deli

last: Hong

first: Coralee

last: Tye

first: Joseph

last: Boyd

first: Jane

last: Lian

first: Gary

last: Stein


title: Evolving Spindlin1 Small Molecule Inhibitors Using Protein Microarrays

city: Memphis

first: JIE

last: Lv

state: TN

address: 262 Danny Thomas Place

department: Department of Computational Biology

institute: St Jude Children's Research Hospital

summary: Using a library of tagged UNC1215 analogs, we screened a protein domain microarray of methyl-lysine effector molecules to rapidly detect compounds with novel binding profiles. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor domain-containing protein Spindlin1 (SPIN1). Structural studies revealed that the symmetric nature of EML405 allows it to simultaneously engage two of SPIN1’s Tudor domains, and also facilitated the rational synthesis of more selective SPIN1 inhibitor (EML631). The EML631 compound engages SPIN1 in cells, blocks its ability to “read” H3K4me3 marks, and inhibits its transcriptional coactivator activity.

overall_design: RNA-seq of control, SPIN1 siRNA knockdown (24 hour post-transfection) and EML631 treated (10 mM, 3 days) T778 cells

first: Narkhyun

last: Bae

first: Donghang

last: Cheng

first: Mark

middle: T

last: bedford

first: Jie

last: Lu

first: Kaifu

last: Chen


title: Hematopoietic Stem/Progenitor Cell Expansion Without Differentiation is Achieved in Zwitterionic Hydrogels

city: Seattle

first: Fabiola

last: Merriam

state: WA

address: 1100 Fairview Ave N

department: Clinical Research

institute: Fred Hutchinson Cancer Research Center

summary: We report the application of Zwitterionic Hydrogel to enable differention-free expansion of human hematopoietic stem/progenitor cells (HSPC) derived from cord blood.

overall_design: To have an advanced insight on the activity of HSPC in  zwitterionic matrix, we conducted mRNA deep-sequencing in HSPC before and after culture

first: Jeff

last: Delrow

first: Ryan

last: Basom


title: Paused RNA polymerase II inhibits new transcriptional initiation

city: Kansas City

first: Julia

last: Zeitlinger

state: MO

address: 1000 E 50th Street

institute: Stowers Institute for Medical Research

type: Genome binding/occupancy profiling by high throughput sequencing

summary: RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. But it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

overall_design: Refer to individual Series


title: Epithelial-Mesenchymal Micro-Niches Govern Stem Cell Lineage Choices

city: New York

first: Hanseul

last: Yang

state: NY

address: 1230 York Avenue

institute: Rockefeller University

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


title: Genome-wide DNA damage response following ionizing radiation

city: Darmstadt

first: Alexander

last: Rapp

address: Schnittspahnstr. 10

country: Germany

institute: Biology

summary: H2AX phosphorylation at Ser139 (gH2AX) is an early key event of the DNA damage response (DDR) following DNA double strand breaks (DSB) induction. Although gH2AX distribution has been extensively used as a damage marker, genome-wide investigation of the subsequent DNA repair has been poorly addressed. Here we present ChIP-Seq-based distributions of Histone H3, H2AX and gH2AX under physiological conditions in HeLa cells. Additionally the same histones were studied after a single exposure to 10 Gy X-ray at 0.5, 3 and 24 hours post irradiation

overall_design: In HeLa cells ChIP-seq data for histone H3, H2AX and gammaH2AX were analysed in unirradiated cells as well as after 0.5, 3 and 24 h. Input samples were included and one technical (Sequencing)  and one biological (irradiation, library prep and sequencing) replicate was done.

first: Wei

last: Yu

first: Francesco

last: Natale

first: M

last: Cardoso


title: Major roles of cyclobutane pyrimidine dimers, nucleotide excision repair and ATR in the alternative splicing response to UV irradiation

city: Barcelona

first: Antonio

last: Torres-Mendez

address: C/ Dr. Aiguader 88

department: Systems Biology

country: Spain

institute: Centre for Genomic Regulation

summary: Alternative splicing (AS) is the main process that amplifies DNA information and is a crucial target of signaling cascades resulting from UV irradiation of human cells. The specific impact of UV-induced cyclobutane pyrimidine dimers (CPDs) at the transcriptional and AS levels has not been addressed. By using a CPD photolyase, a flavoenzyme that directly reverts CPDs, we analyze the contribution of this lesion to the expression program in human keratinocytes.

overall_design: mRNA profiles of human keratinocytes (HaCaT cell line) expressing the CPD photolyase from Potorous tridactylus in three different conditions: 1. Control: Non treated cells and inactive photolyase. 2. UV treated cells: 15 J/m2 (UVC, 254 nm) and inactive photolyase, harvested 6 hours after UV irradiation and 3. Same as before but with an active photolyase due to exposure to white light for 2 hours.

first: Manuel

middle: J

last: Muñoz

first: Nicolás

last: Nieto Moreno

first: Luciana

last: Giono

first: Adrián

last: Cambindo Botto

first: Gwendal

last: Dujardin

first: Giulia

last: Bastianello

first: Stefania

last: Lavore

last: Torres-Méndez

first: Carlos

middle: F

last: Menck

first: Benjamin

last: Blencowe

last: Irimia

first: Marco

last: Foiani

first: Alberto

last: Kornblihtt


title: Epithelial-Mesenchymal Micro-Niches Govern Stem Cell Lineage Choices (SC RNA-Seq)

first: Hanseul

last: Yang

summary: Adult tissue stem cells (SCs) reside in niches, which through intercellular contacts and signaling, influence SC behavior. Once activated, SCs typically give rise to short-lived transit-amplifying cells (TACs), which then progress to differentiate into their lineages. Here, using single cell RNA-sequencing, we unearth unexpected heterogeneity among SCs and TACs of hair follicles. We trace the roots of this heterogeneity to micro-niches along epithelial-mesenchymal interfaces, where progenitors display molecular signatures reflective of spatially distinct local signals and intercellular interactions. Using lineage-tracing, temporal single cell analyses and chromatin landscaping, we show that SC plasticity becomes restricted in a sequentially and spatially choreographed program, culminating in seven spatially arranged uni-lineage progenitors within TACs of mature follicles. By compartmentalizing SCs into micro-niches, tissues gain precise control over morphogenesis and regeneration: Some progenitors specify lineages immediately; others retain potency, preserving self-renewing features established early while progressively restricting lineages as they experience dynamic changes in microenvironment.

overall_design:
Mouse bulge hair follicle stem cell, hair germ, basal transient amplifying cells (TACs) and dermal papilla at the indicated hair cycle were FACS-purified for single cell RNA-seq.

The cell type of each sample is available in the file "GSE90848_cell_type_definitions.txt.gz" on the series record.

first: Elaine

last: Fuchs


title: Inhibition of eukaryotic translation by the antitumor natural product Agelastatin A

city: Baltimore

first: Boris

last: Zinshteyn

state: Maryland

address: 725 N Wolfe St, PCTB 714

department: Molecular Biology and Genetics

institute: Johns Hopkins University School of Medicine

summary: DMS probing of AglA and CHX binding sites on the yeast ribosome

overall_design: Purified ribosomes are probed with DMS to identify drug binding sites.

first: Brandon

last: McClary

first: Melanie

last: Meyer

first: Morgan

last: Jouanneau

first: Simone

last: Pellegrino

first: Gulnara

last: Yusupova

first: Anthony

last: Schuller

first: Jeremy Chris

middle: P

last: Reyes

first: Junyan

last: Lu

first: Zufeng

last: Guo

first: Safiat

last: Ayinde

first: Cheng

last: Luo

first: Yongjun

last: Dang

first: Daniel

last: Romo

first: Marat

last: Yusupov

first: Rachel

last: Green

first: Jun

middle: O

last: Liu


title: Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-Seq]

first: Julia

last: Zeitlinger

overall_design: ChIP-seq was performed against Pol II, TFIIA and TFIIB in Drosophila melanogaster Kc167 cells.

first: Wanqing

last: Shao


title: Cis Regulatory Element Scan by Tiling-deletion and Sequencing

city: La Jolla

first: Bing

last: Ren

state: CA

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

institute: University of California, San Diego School of Medicine

summary: Here we report a highly scalable strategy for unbiased discovery and functional characterization of cis regulatory elements in the genome. These results demonstrate the utility of our cis element discovery strategy and reveal the dual function of gene promoters: they not only initiate transcription of their own genes, but could also control transcription of nearby genes.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
As proof of principle, we focused on the 2Mbp POU5F1 locus (hg19 chr6:30120000-32150000) in hESCs with the goal to identify cis-regulatory elements involved in the transcriptional control of POU5F1. We first designed 11,570 sgRNA pairs that would introduce ~2kb-deletions in a highly nested manner with ~100bp step sizes. As negative controls, we included 424 ineffective oligos lacking the PAM sequence necessary for effective dsDNA breaks. Additionally, we also included 6 sgRNA pairs that target the eGFP coding sequence as positive controls. The dual CRISPR expressing cassettes were cloned into lentiCRISPRv2 vector, and the resulting lentiviral libraries were verified to be of high quality and capable of generating genomic deletions.

ATAC-seq is a widely used method to mapping the open chromatin landscape of the cells. In this study, we perfomed ATAC-seq to compare the open chromain patters in wild-type and two mutant clones with PRRC2A and MSH5 core promoter region deletions. The core promoters of PRRC2A and MSH5 have been identified as distal enhancer of POU5F1 and we want to test if and how the deletion of distal enhancer affect the open chromaitn patterns.

first: Yarui

last: Diao

first: Rongxin

last: Fang

first: Bin

last: Li

first: Zhipeng

last: Meng

first: Ryan

last: Marina

first: Inkyung

last: Jung

first: Kun-Liang

last: Guan


title: Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-nexus spike-in]

overall_design: ChIP-nexus was performed against Pol II in Drosophila melanogaster Kc167 cells. A fix amount of spike-in is added into each sample in order to control for the loss of Pol II ChIP signal after Triptolide treatment or NELF depletion.


title: Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Cgla RNA-seq]

status: Public on Apr 15 2017

city: Iowa City

last: He

email: emptyhb@gmail.com, bin-he@uiowa.edu

state: IA

address: 129 East Jefferson St., Biology Building 450

department: Biology

institute: the University of Iowa

summary: In S. cerevisiae, the phosphate starvation (PHO) responsive transcription factors Pho4 and Pho2 are jointly required for induction of phosphate response genes and survival in phosphate starvation conditions. In the related human commensal and pathogen C. glabrata, Pho4 is required but Pho2 is dispensable for survival in phosphate-limited conditions and is only partially required for inducing the phosphate response genes. This reduced dependence on Pho2 evolved in C. glabrata and closely related species. Pho4 orthologs that are less dependent on Pho2 induce more genes when introduced into the S. cerevisiae background, and Pho4 in C. glabrata both binds to more sites and induces more genes with expanded functional roles compared to Pho4 in S. cerevisiae. We used RNA-seq to profile the transcriptome of wild type and mutants of Pho4 / Pho2 in C. glabrata, to identify genes induced by Pho4.

overall_design: The goal is to identify genes induced by Pho4 in C. glabrata (CgPho4), either with or without C. glabrata Pho2 (CgPho2) in a genetic background where the negative regulator of Pho4, Pho80, is deleted. Pairwise comparisons such as that between PHO4 wild-type and pho4Δ strain, in the pho80Δ CgPHO2 background, will identify genes induced by Pho4 in the presence of Pho2. For each strain, two biological replicates, i.e. the same genotype grown, collected and processed separately, were analyzed by RNA-seq.

middle: Z

last: HE

first: Xu

last: ZHOU

first: Erin

last: O'Shea


title: Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Scer RNA-seq]

summary: In S. cerevisiae, the phosphate starvation (PHO) responsive transcription factors Pho4 and Pho2 are jointly required for induction of phosphate response genes and survival in phosphate starvation conditions. In the related human commensal and pathogen C. glabrata, Pho4 is required but Pho2 is dispensable for survival in phosphate-limited conditions and is only partially required for inducing the phosphate response genes. This reduced dependence on Pho2 evolved in C. glabrata and closely related species. Pho4 orthologs that are less dependent on Pho2 induce more genes when introduced into the S. cerevisiae background, and Pho4 in C. glabrata both binds to more sites and induces more genes with expanded functional roles compared to Pho4 in S. cerevisiae. We used RNA-seq to profile the transcriptome of wild type and mutants of Pho4 / Pho2, or Pho4 ortholog swap in S. cerevisiae, to identify genes induced by Pho4 or its orthologs in S. cerevisiae background.

overall_design: The goal is to identify genes induced by each of the eight Pho4 orthologs in the S. cerevisiae background, either with or without S. cerevisiae Pho2 (ScPho2) and with the negative regulator of Pho4, Pho80, deleted. Pairwise comparisons such as that between a S. cerevisiae strain carrying Pho4 from C. glabrata (CgPho4) and a pho4Δ strain, both in the pho80Δ ScPHO2 background, will identify genes induced by CgPho4 in the presence of ScPho2. For each strain, two biological replicates, i.e. the same genotype grown, collected and processed separately, were analyzed by RNA-seq.

last: K. O'Shea


title: Regulatory landscape fusion in rhabdomyosarcoma by interactions between the PAX3 promoter and FOXO1 regulatory elements

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

institute: CABD/CSIC

summary:
Results: Using a combination of 4C-seq datasets, we were able to model the three-dimensional organisation of the translocated chromosome in a PAX3:FOXO1 fusion-positive alveolar rhabdomyosarcoma cell line. We show that PAX3 and FOXO1 regulatory landscapes fuse into a novel TAD, allowing the PAX3 promoter to interact ectopically with FOXO1 sequences with potential enhancer function. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions.

Conclusions: Our results suggest that the chromosomal translocation that leads to ARMS development generates a novel TAD that favours ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories, which may be an essential step in the tumorigenic process, as expression in a particular cell type, the often elusive cell-of-origin, may be required for disease development.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 4C-seq samples for FoxO1 and Pax3 gene promoters in E9.5 whole mouse embryos, as well as for the PAX3 promoter and several ECRs and CTCF binding sites present at the PAX3:FOXO1 locus in the patient-derived alveolar rhabdomyosarcoma (ARMS) cell line RMS.

first: Cristina

last: Vicente-GarcÃa

first: Barbara

last: Villarejo-Balcells

first: Ibai

last: Irastorza-Azcárate

first: Silvia

last: Naranjo

first: Rafael

middle: D

last: Acemel

first: Peter

last: Rigby

first: Damien

last: Devos

first: JosÃ©

middle: L

first: Jaime

last: Carvajal


title: Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Cgla ChIP-seq]

first: Bin

summary: In S. cerevisiae, the phosphate starvation (PHO) responsive transcription factors Pho4 and Pho2 are jointly required for induction of phosphate response genes and survival in phosphate starvation conditions. In the related human commensal and pathogen C. glabrata, Pho4 is required but Pho2 is dispensable for survival in phosphate-limited conditions and is only partially required for inducing the phosphate response genes. This reduced dependence on Pho2 evolved in C. glabrata and closely related species. Pho4 orthologs that are less dependent on Pho2 induce more genes when introduced into the S. cerevisiae background, and Pho4 in C. glabrata both binds to more sites and induces more genes with expanded functional roles compared to Pho4 in S. cerevisiae. We used Chromatin-ImmunoPrecipitation with exonucleas followed by high-throughput sequencing (BioChIP-seq) to identify the binding locations of Pho4 from both S. cerevisiae and C. glabrata in the S. cerevisiae background lacking the negative regulator Pho80, and either with or without Pho2.

overall_design: We made C. glabrata strains carrying a C-terminally tagged (3x FLAG) version of the endogenous Pho4 either with or without the endogenous Pho2. We also made strains carrying an N-terminally tagged (V5) version of the endogenous Pho2. Mock strains were made to correspond to the experimental strain's genetic background, but lacking the epitope tag. All strains were grown to mid-log phase, washed and released into no phosphate media for 1 hour and harvested for ChIP analysis. We used ChIP-exo, a variation of the ChIP protocol using exonuclease to increase the resolution and signal-to-noise ratio, to query the genomic binding locations of both Pho4 and Pho2 in C. glabrata. A routinely used control for ChIP is the input chromatin for normalizing against heterogeneity in the fragmentation process. This, however, doesn't apply to the ChIP-exo technique. We use either hte mock sample instead for peak finding and generating the WIG files.


title: Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Scer ChIP-seq]

summary: In S. cerevisiae, the phosphate starvation (PHO) responsive transcription factors Pho4 and Pho2 are jointly required for induction of phosphate response genes and survival in phosphate starvation conditions. In the related human commensal and pathogen C. glabrata, Pho4 is required but Pho2 is dispensable for survival in phosphate-limited conditions and is only partially required for inducing the phosphate response genes. This reduced dependence on Pho2 evolved in C. glabrata and closely related species. Pho4 orthologs that are less dependent on Pho2 induce more genes when introduced into the S. cerevisiae background, and Pho4 in C. glabrata both binds to more sites and induces more genes with expanded functional roles compared to Pho4 in S. cerevisiae. We used Biotin-assisted Chromatin-ImmunoPrecipitation followed by high-throughput sequencing (BioChIP-seq) to identify the binding locations of Pho4 from both S. cerevisiae and C. glabrata in the S. cerevisiae background lacking the negative regulator Pho80, and either with or without Pho2.

overall_design: First, we made S. cerevisiae strains carrying an epitope (AVItag) tagged version of Pho4 from either S. cerevisiae (ScPho4) or C. glabrata (CgPho4), in a genetic background where the negative regulator of Pho4, Pho80 is deleted, and either with or without S. cerevisiae Pho2 (ScPho2). A total of four strains were made and subject to the Bio-ChIP profiling, with one biological sample for each strain. In addition, two strains carrying an untagged version of either ScPho4 or CgPho4 in the same S. cerevisiae background  (pho80∆ ScPHO2) were made to serve as mock samples. For both experiment and mock samples, a corresponding "input" sample (sonicated cell lysates prior to immuno-precipitation) was prepared and subject to sequencing.


title: Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-nexus]

first: Julia

last: Zeitlinger

overall_design: ChIP-nexus was performed against Pol II, TBP, TFIIA, TFIIB, TFIIF, TAF2, XPB and NELF-E in Drosophila melanogaster Kc167 cells.

first: Wanqing

last: Shao


title: mTORC1 balances cellular amino acid supply with demand for protein synthesis through post-transcriptional control of ATF4

city: New Haven

first: Carson

last: Thoreen

state: CT

address: 333 Cedar St. SHM B 145

department: Cellular and Molecular Physiology

institute: Yale School of Medicine

summary: The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that is commonly deregulated in human diseases. Here we find that mTORC1 controls a transcriptional program encoding amino acid transporters and metabolic enzymes through a mechanism also used to regulate protein synthesis. Bioinformatic analysis of mTORC1-responsive mRNAs identified a promoter element recognized by activating transcription factor 4 (ATF4), a key effector of the integrated stress response. ATF4 translation is normally induced by phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) through a mechanism that requires upstream open reading frames (uORFs) in the ATF4 5' UTR. mTORC1 also controls ATF4 translation through uORFs, but independent of changes in eIF2α phosphorylation. mTORC1 instead employs the 4E-binding protein (4E-BP) family of translation repressors. These results link mTORC1-regulated demand for protein synthesis with an ATF4-regulated transcriptional program that controls the supply of amino acids to the translation machinery.

overall_design: RNA-seq analysis of wild-type and ATF4-null HEK293T cells treated with Torin 1 or tunicamycin for 6 h, and ribosome profiling analysis of HEK293T cells treated with Torin 1 for 24 h.


title: Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway

last: He

sample_organism:
Saccharomyces cerevisiae
Nakaseomyces glabratus


title: Effects of small molecules on gene expression in mouse nuclear transfer embryos at the 2 cell stage

city: Cambridge

first: Charles

last: Bradshaw

address: Tennis Court Road

department: Gurdon Institute

institute: University of Cambridge

summary: Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. We find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with deionized bovine serum albumin (dBSA), dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). RNA-seq analyses of SCNT embryos at the 2-cell stage revealed that the treatment with TSA, followed by the VC treatment, resulted in the upregulated expression of the previously identified reprogramming-resistant genes. Moreover, the expression of 2-cell-specific retroelements was upregulated by the sequential treatment with TSA and VC. The best condition for reprogramming is to add TSA for 8 hours after nuclear transfer and then SCNT embryos are further cultured with VC for 7 hours.

overall_design: Total RNA-seq of single mouse SCNT embryos treated with trichostatin A and/or vitamin C.

first: Kei

last: Miyamoto


title: Time-series transcriptional profiling of MazF-induced E. coli populations

city: Madison

first: Ophelia

middle: S

last: Venturelli

state: WI

address: 440 Henry Mall, 3204C Biochemical Sciences Building

institute: University of Wisconsin-Madison

summary: Synthetic circuits embedded in host-cells compete with cellular processes for limited intracellular resources. We show how funneling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Together, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.

overall_design: Experimental design included two biological replicates and five time points collected every 2 minutes for 8 minutes total. Data set includes 10 total samples. aTc (5 ng/ml) was administered following the first time point (t=0).


title: AG-221 as an effective and on-target therapy in IDH2 mutant AML

city: Melbourne

first: Lev

last: Kats

state: VIC

address: 305 Grattan Street

country: Australia

institute: Peter MacCallum Cancer Centre

summary: Herein we developed a multigenic AML model with inducible expression of IDH2R140Q and constitutive expression of mutant DNMT3AR882H and NRASG12D. Using genetic de-induction and the first-in-class IDH2 inhibitor AG-221, we demonstrate that despite the presence of multiple oncogenic lesions, AML cells expressing mutant IDH2 depend on continued production of 2-HG to maintain their leukemic potential. Moreover, by comparing pharmacological inhibition and genetic depletion of IDH2R140Q we validate AG-221 as a highly on target inhibitor and identify key downstream pathways including GATA1 that are critical for disease maintenance.

overall_design: RNA sequencing of mutant IDH2 AML cells treated with vehicle, doxycycline (genetic depletion of IDH2R140Q) or AG-221 (IDH2 inhibitor)

first: Ricky

last: Johnstone


title: Single glomerular mesangial cells RNA-seq analysis

city: Nanjing

first: Shaolin

last: Shi

state: Jiangsu

address: 305 East Zhongshan Rd.

department: National Clinical Research Center for Kidney Diseases

institute: Jinling Hospital, Nanjing University School of Medicine

summary: Available single-cell RNA-seq analyses have revealed that individual cells of the same type differ substantially in gene expression. We wonder whether glomerular mesangial cells, the cell type that support the unique structure of glomeruli, also exhibit big difference in gene expression among individual cells; and what biological information could be obtained from the single-mesangial cell RNA-seq data. Therefore, we isolated mouse glomeruli by Dynabead/magnetic concentration method, and digested them with enzymes to dissociate the cells. We loaded the single cell suspension to a Fluidigm C1 Single-Cell Auto Prep System for single cell cDNA preparation. The cDNA samples were analyzed with mesangial cell marker, Gata3,and the Gata3 positive samples were amplified and underwent sequecing using Illumina Highseq 2000 system.

overall_design: In total, 14 single mesangial cells from a C57BL/6 male mouse were analyzed

first: Yuqiu

last: Lu

first: Yuting

last: Ye


title: Transcriptional repression by non-canonical EED stimulation of histone deacetylase activity is required for heart maturation independently of H3K27me3 [RNA-seq for EED and HDAC rescue]

status: Public on Apr 14 2017

city: Chicago

first: Yong

last: Peng

state: IL

address: 929 E 57th Street

department: Department of Chemistry

institute: University of Chicago

summary: Through H3K27me3 and H3K27ac ChIP-seq and RNA-seq data in wile-tpye (WT) and EED-knockout (CKO) mouse cardiomyocytes, we unexpectedly uncovered a novel mechanism of PRC2-mediated gene repression. EED inactivation in the postnatal heart (EEDCKO) caused progressive, lethal dilated cardiomyopathy, with upregulation of components of the slow-twitch muscle gene program. Surprisingly, upregulation of these genes was not associated with their loss of H3K27me3, but rather with their gain of H3K27 acetylation (H3K27ac), an activating histone mark. Moreover, re-expression of EED in juvenile hearts rescued heart function and normalized H3K27ac, but not H3K27me3.

overall_design: RNA-seq in isolated adult cardiomyocytes from 2-month old mice, Wildtype (WT or Het),  cardiac conditional EED Knockout (CKO) and CKO injected with AAV9 expressing luciferase, EED or HDAC1/2..

first: Shanshan

last: Ai

first: Chen

last: Li

first: Fei

last: Gu

first: Qing

last: Ma

first: Xianhong

last: Yu

first: Yanzhu

last: Yue

first: Jinghai

last: Chen

first: Zhiqiang

last: Lin

first: Pingzhu

last: Zhou

first: Huafeng

last: Xie

first: Terence

last: Prendiville

first: Wen

last: Zheng

first: Yuli

last: Liu

first: Stuart

last: Orkin

first: Da-Zhi

last: Wang

first: William

last: Pu

first: Aibin


title: A sister of NANOG regulates genes expressed in pre-implantation human development

status: Public on Apr 16 2017

city: Oxford

first: Thomas

last: Dunwell

address: South Parks Road

department: Zoology

institute: University of Oxford

summary: We examined the ability of human NANOGNB to regulate gene expression by ectopically expressing the gene in human dermal fibroblasts.

overall_design: NANOGNB and an empty control vector were transfected individually to drive ectopic expression in human dermal fibroblasts, in triplicate.

first: Peter

last: Holland


title: Malat1 interacts with Suv39h1 to regulate MyoD transcriptional activity and is down-regulated by miR-181 binding during myogenic differentiation and muscle regeneration

city: Shenzhen

first: Kun

last: Sun

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

department: Cancer Research

institute: Shenzhen Bay Laboratory

summary: As one of the most abundant lncRNAs in various cell types the exact cellular function of Malat1 is a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both cell culture and knock-out mouse, our findings demonstrate that Malat1 plays a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knock-down of Malat1 accelerated the myogenic differentiation in cultured cells. Consistently in vivo Malat1 knock-out mouse displayed enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic Mdx mouse also improved the muscle regeneration. Mechanistically, we showed that in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD binding loci, causing trimethylation of  histone 3 lysine 9 (H3K9me3) which suppresses the target gene expression. Upon differentiation, miR-181a expression increases and targets the nuclear Malat1 transcripts for degradation through Ago2 dependent nRISC machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1β/HDAC1 repressive complex and displacement by a Set7 containing activating complex, which allows MyoD trans-activation to occur. Together our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation.

overall_design: 2 RNA-seq experiments performed

first: Huating

first: Hao

first: Xiaona


title: Transcriptional repression by non-canonical EED stimulation of histone deacetylase activity is required for heart maturation independently of H3K27me3 [RNA-seq]

first: Yong

last: Peng

summary: Through H3K27me3 and H3K27ac ChIP-seq and microarray data in wile-tpye (WT) and EED-knockout (CKO) mouse cardiomyocytes, we unexpectedly uncovered a novel mechanism of PRC2-mediated gene repression. EED inactivation in the postnatal heart (EEDCKO) caused progressive, lethal dilated cardiomyopathy, with upregulation of components of the slow-twitch muscle gene program. Surprisingly, upregulation of these genes was not associated with their loss of H3K27me3, but rather with their gain of H3K27 acetylation (H3K27ac), an activating histone mark. Moreover, re-expression of EED in juvenile hearts rescued heart function and normalized H3K27ac, but not H3K27me3.

overall_design: RNA-seq in isolated adult cardiomyocytes from 2-month old mice, Wildtype (WT) cardiac conditional EED Knockout (CKO).

first: Shanshan

last: Ai

first: Chen

last: Li

first: Fei

last: Gu

first: Qing

first: Xianhong

first: Yanzhu

first: Jinghai

last: He


title: Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition.

first: giovanni

last: iacono

state: Spain

address: Baldiri Reixac, 4

department: single cell sequencing

institute: CNAG,Centro Nacional de Análisis Genómico

summary: Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.

overall_design: anti-H3k4me3 ChIP-seq; 12 samples. 3 Kansl1 +/- , 3 Del/+, 6 wts

last: Arbogast

first: Giovanni

last: Iacono

first: Claire

last: Chevalier

first: Bert

last: De Vries

first: Yann

last: Herault


title: EED orchestration of heart maturation through interaction with HDACs is H3K27me3-independent

summary: In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have been performed, gene repression is associated with PRC2 trimethylation of H3K27 (H3K27me3). However, it is uncertain whether PCR2 writing of H3K27me3 is mechanistically required for gene silencing. Here we studied PRC2 function in postnatal mouse cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after each cell cycle. EED (Embryonic Ectoderm Development) inactivation in the postnatal heart (Eed CKO ) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in Eed CKO was not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. HDAC overexpression normalized Eed CKO heart function and expression of derepressed genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive mechanism that is essential for normal heart function. Our results further illustrate that organ dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring.

overall_design: Refer to individual Series


title: Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse.

summary: Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1-/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.

overall_design: 6 samples RNA-seq. 3 kos, 3wts.

first: Dévina

last: Ung

first: Hamid

last: Méziane

first: Henk

middle: G

last: Stunnenberg

first: John

middle: B

last: Vincent

first: Nael

middle: N

last: Kasri

last: Hérault

first: Frédéric

last: Laumonnier


title: Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance [ATAC-seq]

status: Public on Apr 13 2017

city: Philadelphia

first: Sydney

middle: M

last: Shaffer

state: PA

address: BRB II/III, Room 445, 421 Curie Blvd

institute: University of Pennsylvania

summary: Therapies targeting signaling molecules mutated in cancers can often have striking short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can sometimes result from a secondary mutations in rare cells, but other times, there is no clear genetic cause, raising leaving the possibility of non-genetic rare cell variability. Here, we show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug treatment. This variability involves semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces an epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins withis a progressive process consisting of a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general rare-cell expression program.

overall_design: We performed FACS to isolate EGFR-high populations of WM989 melanoma cells at three time points (untreated, 1 week in vemurafenib, 4 weeks in vemurafenib) for RNA sequencing and ATAC sequencing. Each sample has three biological replicates.


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of 10 enhancers in combination

city: Dallas

first: Gary

middle: Chung

last: Hon

state: TX

address: 5323 Harry Hines Blvd.

department: OB/GYN

institute: UT Southwestern

summary: Transcriptional enhancers orchestrate cell-type specific gene expression programs critical to eukaryotic development, physiology, and disease. However, despite the large number of enhancers now identified, only a small number have been functionally assessed. Here, we develop MOsaic Single-cell Analysis by Indexed CRISPR Sequencing (Mosaic-seq), a method that measures one direct phenotype of enhancer repression: change of the transcriptome, at the single cell level. Using dCas9-KRAB to suppress enhancer function, we first implement a multiplexed system to allow the simultaneous measurement of the transcriptome and detection of sgRNAs by single cell RNA sequencing. We validate this approach by targeting the HS2 enhancer in the well-studied beta-globin locus. Next, through computational simulation, we demonstrate strategies to robustly detect changes in gene expression in these single cell measurements. Finally, we use Mosaic-seq to target 71 hypersensitive regions belonging to 15 super-enhancers in K562 cells by utilizing a lentiviral library containing 241 unique-barcoded sgRNAs. Our results demonstrate that Mosaic-seq is a reliable approach to study enhancer function in single cells in a high-throughput manner.

overall_design: The overall design consists of RNA sequencing in bulk and in single K562 cells. As a control, we performed single-cell RNA sequencing of K562 cells expressing dCas9-KRAB and a control sgRNA targeting the non-expressed HSBP1 gene in two biological replicates. As a pilot project, we performed single cell RNA-Seq of dCas9-KRAB K562 cells after pooled infection of a library of 10 sgRNA viruses targeting promoters and enhancers in the beta-globin locus. As controls for this experiment, we individually infected these 10 sgRNAs and either performed 1) bulk RNA sequencing or 2) single cell RNA sequencing after pooling the separately infected cells. Finally, we scaled this single-cell approach to measure transcriptome changes for a library of 241 sgRNAs spanning 71 hypersensitive sites from 15 super-enhancers in K562 cells. The data in this GEO series relate to the application of Mosaic-Seq for combinatorial repression of enhancers. Four experiments are performed with low MOI, and one is performed at high MOI.

first: Shiqi

last: Xie


title: Cooperativity between EMT and non-EMT cells promotes breast cancer metastasis via paracrine GLI activation

city: Houston

first: Edward

middle: S.

last: Chen

address: 1 Baylor Plz

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: Recent fate mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that it failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumor cells in a paracrine manner in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI-926, a Smoothened inhibitor, blocks this effect, and inhibits growth in PDX models. In human breast tumors, the EMT-TFs strongly correlate with activated Hedgehog/GLI signaling, but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis in part via non-cell autonomous effects that activate the Hh pathway. While all Hh inhibitors may act against tumors with canonical Hh/GLI signaling, only GLI inhibitors would act against EMT-induced GLI activation. In recent years, immunohistochemical analyses and multiplex high-throughput single cell sequencing of human tumor cells have shown that tumors are composed of diverse cell subpopulations containing different driver mutations, gene and protein expression profiles, growth rates, and responses to chemotherapeutics. Such heterogeneity is exacerbated by cellular plasticity, where some cells may undergo oncogenic epithelial-to-mesenchymal transition (EMT), resulting in loss of cell-cell adhesion and polarity, reduced epithelial and elevated mesenchymal protein expression, increased migration and invasion, and enhanced dissemination from the primary tumor. Because metastases in patients appear epithelial, the reverse process, mesenchymal-to-epithelial transition (MET), may occur to allow tumor cell colonization in secondary metastatic sites, establishing cellular plasticity as an important aspect of tumor progression. However, the role of EMT in carcinoma metastasis is controversial. Recent lineage tracing studies argue against the requirement of EMT for metastasis, as reporter-tagged cells that underwent a previous EMT were not found at the secondary site. However, these studies did not address the potential cooperation between EMT and non-EMT cells during the metastatic process, as EMT cancer cells may enable non-EMT cells to gain access to the secondary site, leading to macrometastatic growth. Thus, metastasis can be influenced by intratumoral heterogeneity: where a small proportion of primary tumor cells that have undergone an EMT may influence neighboring, non-EMT tumor cells. Twist1, Snail1, and Six1 are EMT-inducing transcription factors (EMT-TFs) that have all been associated with breast cancer metastasis. All three EMT-TFs regulate critical developmental processes such as cell survival, migration and invasion; in part by influencing EMT. In addition, they are downregulated post-embryogenesis, but re-expressed in various cancers where they cell autonomously induce EMT, resulting in increased percentages of tumor initiating cells (TICs) and enhanced metastasis. In carcinomas, Twist1 and Snail1 transcriptionally repress E-Cadherin (E- Cad) and upregulate mesenchymal genes. Similarly, Six1 overexpression induces EMT by regulating E-Cad localization and altering other EMT markers. During development and cancer, EMT-TFs act in concert with several signaling networks including TGFβ, Wnt, and Hedghog (Hh). The Hedgehog (Hh) signaling pathway is a prominent regulator of embryonic development, where Hh ligands function as morphogens to control numerous developmental processes. Interestingly, in Drosophila eye development, hh is a direct target of sine oculis (the homolog of Six1) and Six1 regulates Hh/GLI signaling during lung development and in fibroblasts. Additionally, Twist1 and Hh/GLI signaling are intimately linked during development, and recently Twist1 and Snail1 were associated with the Hh pathway in TICs. In mammals, canonical activation of Hh/GLI signaling involves binding of one the Hh ligands such as(either, Desert (DHH), Indian (IHH), or Sonic Hedgehog (SHH) to Patched-1 (PTCH1) or Patched-2 (PTCH2) receptors, relieving the inhibitory activity of PTCH on Smoothened (SMO). When inhibition is relieved, levels of the transcriptional activator forms of one or more GLI transcription factors (GLI1, 2, or 3) increase in the nucleus, resulting in activation of Hh target genes 12. Non-canonical activation of the GLI TFs can occur in a Hh- or SMO-independent manner via secreted factors like TGF-β19. Importantly, autocrine and paracrine Hh-mediated crosstalk between tumor cells and the tumor microenvironment 20 results in increased proliferation, stem cell self-renewal and metastasis in various cancers 21. In basal cell carcinoma (BCC) and medulloblastoma, activated Hh signaling is often due to mutations in pathway components such as PTCH and SMO, while in other tumor types including breast, mutations are not observed at high frequency. Instead there is evidence for Hh ligand-dependent pathway hyperactivity. Because numerous studies link Hh signaling to progression in multiple tumor types, derivatives of cyclopamine (e.g. GDC-0449 and IPI926), a naturally occurring plant-derived steroidal alkaloid which targets SMO, are in clinical trials for select patients with BCC and medulloblastoma with promising efficacy. However, these inhibitors have not shown promise in breast cancer despite evidence for activation of this pathway. Herein, we demonstrate that EMT-TFs Twist1, Snail1 and Six1 influence neighboring carcinoma cells in a non-cell autonomous manner, by increasing EMT features and aggressive properties of cells not expressing these TFs. Six1 is a key mediator of the non-cell autonomous effects downstream of Twist1 and Snail1, and can induce metastasis of non-Six1, non-EMT cells. All three EMT-TFs function non-cell autonomously by activation of GLI-mediated transcription in non-EMT cells, but employ different mechanisms of pathway activation, some of which are Hh ligand and SMO-independent. We find that pharmacological inhibition of GLI, but not SMO, in the non-EMT cells efficiently inhibits the non-cell autonomous phenotypes imparted by all three EMT-TFs. Importantly, we demonstrate that in selected patient derived breast cancer xenograft (PDX) models endogenously expressing high levels of these EMT-TFs (but not Hh ligands), GANT61 (a GLI1/2 inhibitor) inhibits tumor growth whereas IPI926 (a SMO inhibitor) did not. Our data suggest that upstream SMO inhibitors may not prove efficacious in tumors where a proportion of cells activate GLI-TF via EMT-TFs, and instead argue that inhibitors directly targeting GLI may be more effective.

overall_design: Human breast cancer xenograft RNA sequencing. Mice were untreated.

first: Michael

last: Lewis

first: Deepika

last: Neelakantan

middle: U

last: Oliphant

first: Xiaomei

last: Zhang

first: Lukas

last: Simon

first: David

last: Henke

first: Chad

middle: A

last: Shaw

first: Meng

last: Wu

first: Susan

last: Hilsenbeck

first: Lisa

last: White

first: Hengbo

last: Zhou

first: Heide

last: Ford


title: H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells [RNA-seq]

city: Detroit

first: Benjamin

last: Kidder

state: MI

address: 4100 John R St

department: Oncology

institute: Wayne State University

summary: Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell self-renewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these ASEs. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells.

overall_design: RNA-Seq of murine shLuc and shKdm5b ES cells.


title: The 3’-UTR of MYC couples RNA polymerase II function to ribonucleotide levels

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary: Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on an external supply of glutamine, an observation termed “glutamine addiction”. Surprisingly, human colon cancer cells, which express high levels of MYC due to mutations in the WNT pathway, are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses endogenous MYC expression via the 3’-UTR of the MYC mRNA, enabling escape from apoptosis. The glutamine-dependent metabolites that mediate this regulation are adenosine nucleotides. Glutamine deprivation causes a global reduction in promoter association of RNA Polymerase II (RNAPII) and reduces the RNAPII traveling ratio, indicative of defects in transcriptional elongation. While RNAPII function is restored by activation of MYC on most genes, restoration of elongation is imperfect and activation of MYC in the absence of glutamine enhances stalling of RNAPII in the body of individual genes, promoting the formation of R-loops. Since stalling of RNAPII and R-loop formation are potential causes of DNA damage, our data argue that the MYC 3’-UTR has a critical role in maintaining genome instability when ribonucleotide levels are low.

overall_design: RNAPII binding in glutamine starved HCT116 cells and ectopic expression of MYC.

first: Francesca

middle: R

last: Dejure

first: Nadine

last: Royla

first: Steffi

last: Herold

first: Jacqueline

last: Kalb

first: Susanne

last: Walz

first: Carsten

last: Ade

first: Guido

last: Mastrobuoni

first: Jens

last: Vanselow

first: Andreas

last: Schlosser

first: Elmar

last: Wolf

first: Stefan

last: Kempa


title: Directed differentiation of human pluripotent stem cells to microglia

city: New York

first: Panagiotis

last: Douvaras

state: New York

address: 619 West 54th Street

institute: The New York Stem Cell Foundation

summary: Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the central nervous system, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell (PSC) lines.

overall_design: Comparison of human pluripotent stem cell derived microglia to human primary microglia, human primary hepatic macrophages and peripheral blood derived macrophages non-polarized or polarized with IFN-γ and LPS, IL-4 and IL-13 or IL-10.

first: Bruce

last: Sun

first: Minghui

last: Wang

first: Ilya

last: Kruglikov

first: Gregory

last: Lallos

first: Matthew

last: Zimmer

first: Cecile

last: Terrenoire

first: Bin

last: Zhang

first: Sam

last: Gandy

first: Eric

last: Schadt

first: Donald

last: Freytes

first: Scott

last: Noggle

first: Valentina

last: Fossati


title: Profiling DNA Replication Timing in Zebrafish Embryos Reveals Dynamic Regulation During Development

city: Oklahoma

first: Christopher

middle: Lee

last: Sansam

state: OK

address: 825 NE 13th Street

department: Cell Cycle and Cancer Biology Program

institute: Oklahoma Medical Research Foundation

summary: The temporal order of DNA replication is modified during differentiation, but when a replication timing program is established and what alterations occur in vivo during embryogenesis are not known.  Here we used zebrafish embryos to generate genome-wide, high-resolution replication timing maps throughout development. Unexpectedly, a non-random and defined replication timing program was evident in the rapid cell cycles before the midblastula transition. The majority of the genome undergoes dynamic shifts in replication timing throughout development as the timing program is decompressed, with many abrupt timing changes occurring during lineage specification. Strikingly, the long arm of chromosome 4 undergoes a developmentally regulated switch to late replication, reminiscent of mammalian X chromosome inactivation. This analysis also revealed a strong relationship between early replication and epigenetic marks at enhancers. Collectively, these data reveal the major changes in replication timing that occur during zebrafish embryogenesis, and demonstrate its dynamic regulation during vertebrate development.

overall_design: Examination of DNA replication timing at six developmental timepoints

first: Joseph

middle: C

last: Siefert


title: Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance

first: Sydney

last: Shaffer

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


title: Gene expression signature of vemurafenib resistance in WM989 and WM983B melanoma cells

overall_design: We performed RNA sequencing on WM989 cells without vemurafenib, after 48 hours of treatment, and upon the development of resistance.


title: H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells [ChIP-seq]

first: Benjamin

last: Kidder

overall_design: ChIP-Seq for RNAPII, RNAPII-Ser5P, RNAPII-Ser2P in murine shLuc and shKdm5b ES cells.


title: Translation initiation from conserved non-AUG codons provides additional layers of regulation and coding capacity

city: Cork

first: Audrey

last: Michel

state: Cork

address: Western Gateway, Western Road

department: School of Biochemistry and Cell Biology

country: Ireland

institute: University College Cork

summary: Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700 nt 5’-leader and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell-free extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4 in controlling translation. We also found that the 5’ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5’ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro. In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscore the emerging significance of non-canonical initiation events in controlling gene expression.

overall_design: Ribosome profiling of  N. crassa wild type cells and N. crassa cells grown in the presence or absence of 3-Amino-1,2,4-triazole (3AT)

first: Ivaylo

middle: P

last: Ivanov

first: Jiajie

last: Wei

first: Stephen

last: Caster

first: Kristina

last: Smith

first: Ying

first: Michael

last: Freitag

first: Jay

last: Dunlap

first: Deborah

last: Bell-Pedersen

first: John

middle: F

last: Atkins

last: Sachs


title: Transcriptome-wide identification of CELF2 functional targets in T cells

first: Kristen

last: Lynch

state: Pennsylvania

address: 906 Stellar Chance Labs 422 Curie Blvd

department: Biochemistry and Molecular Biophysics

summary: CELF2 is a critical regulator of alternative splicing during T cell development and during stimulation-induced T cell activation. Here we utilize RNA-seq to globally profile the functional targets of CELF2 in a monoclonal Jurkat T cell line (JSL1 cells), including transcriptome-wide splicing targets and genes regulated at other levels of mRNA processing

overall_design: We performed strand-specific RNA-seq on JSL1 cells that were unstimulated or stimulated with the phorbol ester PMA, as well as cells in both of these conditions that were depleted for CELF2 through stable lentiviral transfection of shRNAs in duplicate

first: Matthew

last: Gazzara

middle: J

last: Mallory

middle: W

first: Yoseph

last: Barash


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of beta-globin locus (pooled infection)

first: Gary

last: Hon

overall_design: The overall design consists of RNA sequencing in bulk and in single K562 cells. As a control, we performed single-cell RNA sequencing of K562 cells expressing dCas9-KRAB and a control sgRNA targeting the non-expressed HSBP1 gene in two biological replicates. As a pilot project, we performed single cell RNA-Seq of dCas9-KRAB K562 cells after pooled infection of a library of 10 sgRNA viruses targeting promoters and enhancers in the beta-globin locus. As controls for this experiment, we individually infected these 10 sgRNAs and either performed 1) bulk RNA sequencing or 2) single cell RNA sequencing after pooling the separately infected cells. Finally, we scaled this single-cell approach to measure transcriptome changes for a library of 241 sgRNAs spanning 71 hypersensitive sites from 15 super-enhancers in K562 cells.

first: Hon

last: Gary

first: Xie

last: Shiqi


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq TNF-alpha treatment versus mock

first: Shiqi

last: Xie


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of 15 SEs, 71 HSs, 241 sgRNAs


title: Transcriptional repression by non-canonical EED stimulation of histone deacetylase activity is required for heart maturation independently of H3K27me3 [ChIP-seq]

first: Yong

last: Peng

overall_design: RNA-seq and  Profile H3K27ac, H3K27me3, EED and HDAC2 occupancy in isolated adult cardiomyocytes from 2-month old mice, Wildtype (WT) cardiac conditional EED Knockout (CKO) and CKO injected with AAV9 expressing luciferase or HDAC1/2.

first: Shanshan

last: Ai

first: Fei

last: Gu

first: Qing

last: Ma

first: Yanzhu

last: Yue

first: Jinghai

last: Chen

first: Zhiqiang

last: Lin

first: Pingzhu

last: Zhou

first: Huafeng

first: Terence

last: Prendiville

first: Stuart

middle: H

last: Orkin

first: Da-Zhi

last: Wang

first: William

middle: T

last: Pu

first: Aibin

last: He


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells

first: Gary

last: Hon


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Bulk RNA-Seq validation


city: Pittsburgh

first: Cecilia

last: Lo

address: 530 45th St., 8120 Rangos Research Center

department: Developmental Biology

institute: University of Pittsburgh

summary:
Congenital heart disease (CHD) is a prevalent birth defect affecting up to 1% of live births.  While a genetic etiology is indicated by increased recurrence risk and association with chromosomal abnormalities, the genetics of CHD is poorly understood and likely complex.  Here we show hypoplastic left heart syndrome (HLHS), a severe CHD with high morbidity/mortality, has a multigenic etiology.  Analysis of 8 HLHS mutant mouse lines recovered from a large-scale mutagenesis screen showed no mutations are shared in common. In the *Ohia* mouse line, mutations in 2 genes, *Pcdha9*/*Sap130*, act synergistically to cause HLHS. Cardiomyocyte proliferation and differentiation defects likely act in concert with valve abnormalities to cause HLHS.  Network analysis of mutations recovered in HLHS mice and 68 HLHS patients point to Notch signaling disruption, a pathway previously implicated in HLHS. These findings demonstrate large-scale mutagenesis is an effective systems approach for interrogating the complex genetics of human diseases.

SUBMITTER_CITATION:
Journal:  Nature Genetics
Title: The complex genetics of hypoplastic left heart syndrome
Author names: Xiaoqin Liu1, Hisato Yagi1, Shazina Saeed1, Abha S. Bais1, George C. Gabriel1, Zhaohan Chen1, Kevin  A. Peterson4, You Li1, Molly C. Schwartz1, William T. Reynolds1, Manush Saydmohammed1, Brian Gibbs1, Yijen Wu1, William Devine1, Bishwanath Chatterjee1, Nikolai T. Klena1, Dennis Kostka1, Karen L. de Mesy Bentley5, Madhavi K.Ganapathiraju2, Phillip Dexheimer11, Linda Leatherbury7, Omar Khalifa1, Anchit Bhagat1, Maliha Zahid1, William Pu8, Simon Watkins3, Paul Grossfeld9, Stephen Murray4, George A. Porter, Jr.6, Michael Tsang1, Lisa J.Martin10, D.Woodrow.Benson12, Bruce J. Aronow11, Cecilia W. Lo1


title: H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells

first: Benjamin

middle: L

last: Kidder


title: Olfactory stem cell differentiation: horizontal basal cell (HBC) lineage

city: Berkeley

first: Diya

last: Das

state: California

address: 265 Life Sciences Addition #3200

department: Molecular & Cell Biology

institute: University of California, Berkeley

summary: The lineage of the horizontal basal cells (HBC) stem cells and other Sox2eGFP-positive cells from the olfactory epithelium were profiled by single-cell RNA-Seq to identify differentiated cells types, intermediate stages, transition states, and to infer the lineage trajectories.

overall_design: Horizontal basal cell (HBC) stem cells from the olfactory epithelium that were either wild-type or mutant for the transcription factor Trp63/p63 were lineage traced, collected by FACS, and profiled by single-cell RNA-seq.  Additionally, Sox2eGFP transgenic cells from the olfactory epithelium were combined with this data into one data set that was processed together. A minimum of two biological replicates were collected for each time-point/experimental condition. A total of 680 YFP-positive lineage traced cells plus 169 Sox2eGFP-positive cells were used in this analysis.

first: Russell

middle: B

last: Fletcher

first: Levi

last: Gadye

first: Kelly

last: Street

first: Ariane

last: Baudhuin

first: Allon

last: Wagner

first: Michael

last: Cole

first: Quetzal

last: Flores

first: Yoon

middle: G

last: Choi

first: Nir

last: Yosef

first: Elizabeth

last: Purdom

first: Sandrine

last: Dudoit

first: Davide

last: Risso

first: John

last: Ngai


title: Pathogenesis of Hypoplastic Left Heart Syndrome [RNA-seq]

summary: Congenital heart disease (CHD) is a prevalent birth defect affecting up to 1% of live births.  While a genetic etiology is indicated by increased recurrence risk and association with chromosomal abnormalities, the genetics of CHD is poorly understood and likely complex.  Here we show hypoplastic left heart syndrome (HLHS), a severe CHD with high morbidity/mortality, has a multigenic etiology.  Analysis of 8 HLHS mutant mouse lines recovered from a large-scale mutagenesis screen showed no mutations are shared in common. In the *Ohia* mouse line, mutations in 2 genes, *Pcdha9*/*Sap130*, act synergistically to cause HLHS. Cardiomyocyte proliferation and differentiation defects likely act in concert with valve abnormalities to cause HLHS.  Network analysis of mutations recovered in HLHS mice and 68 HLHS patients point to Notch signaling disruption, a pathway previously implicated in HLHS. These findings demonstrate large-scale mutagenesis is an effective systems approach for interrogating the complex genetics of human diseases.

overall_design: Expression profiling using RNA-seq of 16 samples collected from both left ventricle (LV) and right ventricle (RV) of HLHS mice (n=1 for E13.5, E14.5 and E17.5) and their littermate controls (n=1 for E13.5, and n=2 for E14.5 and E17.5 each).

first: Abha

middle: S

last: Bais


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of beta-globin locus (separate infection)

first: Gary

middle: Chung

last: Hon

first: Hon

last: Gary

first: Xie

last: Shiqi


title: Characterization of small RNAs and their targets in Lactuca sativa

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

summary: The small RNAs and their targets were characterized in lettuce (Lactuca sativa) genome by deep sequencing the small RNA populations of leaf tissues (cv. Salinas, Cobham and Diana), inoculated with Bremia and mock. The small RNA targets were also validated using PARE/degradome data derived from the same tissues.

overall_design: We generated integrative maps of small RNA profiles (sRNA) and degradome (PARE) profiles to characterize the miRNAs and their targets from lettuce.

first: Siwaret

last: Arikit

first: Xia

last: Rui


title: Testing mAbs generated by the PCRP pipeline in ChIP-seq application

status: Public on Apr 12 2017

city: Baltimore

first: Anand

last: Venkataraman

state: MD

address: 733 N Broadway, MRB 322

department: Neuroscience

institute: Johns Hopkins University

summary: We report the applicability of multiple PCRP mAbs in ChIP-seq applications in human cell-lines using standard ENCODE protocols.

overall_design: mAbs were tested in 1-4 ENCODE's default human cell-lines. Input control for each mAb and biological replicates for some mAbs are included.

first: Kun

last: Yang

first: Seth

last: Blackshaw

first: Mark

last: Mackiewicz

first: Richard

middle: M

last: Myers


title: AHR ChIP-Seq of female mouse liver following 2 hour TCDD exposure

city: East Lansing

first: Rance

last: Nault

state: United States

address: 1355 Bogue St. B403

department: Department of Pharmacology and Toxicology

institute: Michigan State University

summary: Aryl hydrocarbon receptor ChIP-Seq performed in livers of female mice gavaged with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2hrs

overall_design: Five biological replicates

first: Kelly

middle: A

last: Fader

first: Shaimaa

last: Ahmed

first: Jason

last: Matthews

first: Timothy

last: Zacharewski


title: RNA-seq of overgrown interscapular adipose tissue with biallelic MFN2 mutations

city: Cambridge

first: David

last: Bulger

address: Box 289, Addenbrooke's Hospital

department: Institute of Metabolic Science

institute: University of Cambridge

summary: Mitofusin 2 (MFN2) mutations previously associated with axonal neuropathy have recently been found in patients with upper body overgrowth of adipose tissue. RNA-seq was performed on two of these patients with biallelic MFN2 mutations. The transcriptome demonstrated increased mitochondrial stress signaling, increased cell survive and decreased cell death signatures, and increased protein synthesis.

overall_design: RNAseq of interscapular subcutaneous adipose tissue from two patients with MSL (3 samples each) and five otherwise healthy volunteers (2-3 samples each for a total of 14 control samples) undergoing corrective surgery for idiopathic scoliosis.

first: Brian

last: Lam


title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Human/mouse mixing Drop-Seq experiments for K562 and MEFs

first: Gary

last: Hon

first: Hon

last: Gary


title: Genome reorganisation and transcriptional changes during activation of the human T-cell line Jurkat

city: Edinburgh

first: Jose

middle: Ignacio

last: de las Heras

address: Max Born Crescent

department: Wellcome Trust Centre

institute: University of Edinburgh

overall_design:
Refer to individual Series

Please note that the 170215_GEO_delasHeras_superseries_Jurkat.xls (linked below) contains two spreadsheets: a table, and an explanation of each header of the table.
This table combines transcriptome data with DamID data in a gene-centric manner and forms the basis of this study's conclusion.


title: Oncogenic PIK3CA(H1047R) and CTNNB1(stab) in intestinal organoids

city: Berlin

first: Markus

last: Morkel

address: Charitéplatz 1

department: Dept. of Pathology

institute: Charité - Universitätsmedizin Berlin

summary: Goals of the study was to compare transcripional and phenotypic response of mouse intestinal organoid cultures to the PIK3CA(H1047R) and CTNNB1(stab) oncogenes.

overall_design:
Two biological  replicates of organoids with transgenic tdTomato-Luciferase, tdTomato-PIK3CAH1047R, tdTomato-CTNNB1stab or td-Tomato-PIK3CAH1047R-CTNNB1stab were analysed by RNA-Seq
By comparing 7-10 x 10E7 50bp paired end reads per library we identify transcriptional alterations in the intestinal epithelium following expression of each or both oncogenes,

first: Pamela

last: Riemer


title: GRHL3 binding and the enhancer landscape are reorganized during transitions between different functional states of epidermal keratinocytes

city: Irvine

first: Bogi

last: Andersen

state: CA

address: 839 Health Sciences Dr.

department: Medicine

institute: University of California, Irvine

overall_design: Refer to individual Series


title: Accumulation of RNA on chromatin disrupts heterochromatic silencing

city: Munich

first: Mario

last: Halic

department: Biochemistry - Gene Center

institute: Ludwig-Maximilians-Universität

summary: Long non-coding RNAs (lncRNAs) play a conserved role in regulating gene expression, chromatin dynamics and cell differentiation. They serve as a platform for RNA interference (RNAi)-mediated heterochromatin formation or DNA methylation in many eukaryotic organisms. We found in Schizosaccharomyces pombe, that heterochromatin is lost at transcribed regions in absence of RNA degradation, although establishment is not affected. We show that heterochromatic RNAs accumulate on chromatin, form R-loops and need to be degraded by the Ccr4-Not complex or RNAi to maintain heterochromatic silencing. The Ccr4-Not complex is localized to chromatin independently of H3K9me and degrades chromatin associated transcripts, which is required for transcriptional silencing. Overexpression of heterochromatic lncRNA at a heterochromatic locus abolishes silencing of an ade6 reporter in wild type cells. Furthermore, euchromatic lncRNA accumulate on chromatin and this regulates their transcription. Our results demonstrate that chromatin bound RNA interfere with heterochromatin organization and silencing.

overall_design: RNA, siRNA, ChIP and RIP sequencing in wild type cells and different mutants of S.pombe

first: C

last: Brönner

first: L

last: Salvi

first: I

last: Ugolini

first: M


title: Changes in chromatin association with the nuclear periphery in resting and activated Jurkat T-cells [DamID-seq]

summary: Activation of T-cells induces dramatic changes in genome organisation and gene transcription. Here we identify changes in chromatin association with the periphery by DamID.

overall_design: Maps of genomic regions in proximity to the nuclear lamina were determined in resting and activated Jurkat T-cells using Lamin B1 DamID.

middle: I

first: Michael

last: Robson

first: Eric

last: Schirmer


title: small RNA-Seq of WT, snrk2.2/3/6, and hyl1-2

city: West Lafayette

first: Jian-Kang

last: Zhu

state: IN

address: 625 Agriculture Mall Dr.

department: Department of Horticulture and Landscape Architecture

institute: Purdue University

summary: Using small RNA-Seq(sRNA-Seq) to get miRNA profiling of  WT,  snrk2.2/3/6, and hyl1-2 mutant

overall_design: 3 samples examined: WT, hyl1-2 single mutant,  and snrk2.2/3/6 triple mutant. Each sample has two replicates.

first: Jun

last: Yan

first: Kai

last: Tang


title: Toxicogenomic module associations with pathogenesis: A network based approach to understanding drug toxicity (ChIP-Seq)

city: Indianapolis

first: Keith

last: Goldstein

address: Lilly Corporate Center

department: Investigative Toxicology

institute: Eli Lilly & Co.

summary: Despite investment in toxicogenomics, nonclinical safety studies are still used to predict clinical liabilities for new drug candidates. Network-based approaches for genomic analysis help overcome challenges with whole-genome transcriptional profiling using limited numbers of treatments for phenotypes of interest. Herein, we apply co-expression network analysis to safety assessment using rat liver gene expression data to define 415 modules, exhibiting unique transcriptional control, organized in a visual representation of the transcriptome (the ‘TXG-MAP’). Accounting for the overall transcriptional activity resulting from treatment, we explain mechanisms of toxicity and predict distinct toxicity phenotypes using module associations. We demonstrate that early network responses compliment traditional histology-based assessment in predicting outcomes for longer studies and identify a novel mechanism of hepatotoxicity involving endoplasmic reticulum stress and Nrf2 activation. Module-based molecular subtypes of cholestatic injury derived using rat translate to human. Moreover, compared to gene-level analysis alone, combining module and gene-level analysis performed in sequence identifies significantly more phenotype-gene associations, including established and novel biomarkers of liver injury.

overall_design: Evaluated livers from animals 1, 5, 14 days after bile duct ligation were compared with sham surgery animals. Performed ChIP-seq to investigate 4 transcription factors- cJun, Sp1, HNF and FXR.

first: Jeffrey

last: Sutherland

first: Yue

last: Webster

last: Willy

first: George

last: Searfoss

first: Armando

middle: R

last: Irizarry

first: David

last: Hall

first: James

last: Stevens


title: Transcriptome changes in mouse livers upon Bmal1 or Nr1d1 knock out and interactome of lnc-Crot by 4C-seq

city: San Francisco

first: Zenghua

last: Fan

address: 1550 4th Street

institute: University of California, San Francisco

summary: To explore the circadian regulations of Bmal1, we examined the transcriptome changes in mouse livers upon Bmal1 knock out at two circadian time points, CT0 and CT12. To explore the circadian regulations of Nr1d1, we examined the transcriptome changes in mouse livers upon Nr1d1 knock out at two circadian time points, CT0 and CT12. To explore interactome of lnc-Crot, 4C-seq was performed with lnc-Crot as bait region at CT6 and CT18.

overall_design: RNA-seq of WT and KO at CT0 and CT12. 4C-seq at CT6 and CT18.


title: GRHL3 regulates the chromatin state during epidermal differentiation

first: Bogi

last: Andersen

summary: The process of differentiation is tightly controlled, and such complex coordination of gene expression requires many layers of regulation. One such mechanism of regulation occurs through distal genomic regions called enhancers, which are believed to act as concentrating sites for transcription factors, like GRHL3, and other regulators, forming loops to contact promoters and enhance transcription. Active enhancers have been shown to have high levels of H3K4me1 and H3K27ac modified nucleosomes, with low levels of H3K4me3, while poised developmental enhancers have been shown to have H3K27me3 in place of H3K27ac. Additionally, in each cell type, a select few enhancers have been found to be unusually long (more than 12.5kb) and show high intensity of transcription factor binding and chromatin enhancer signature marks. These regions, termed super enhancers, are frequently tissue-specific and are thought to regulate the unique gene expression programs in each tissue. ChIP-Seq analysis revealed GRHL3 binding to be strongly enriched within enhancers and super enhancers in differentiating keratinocytes, however the effect of GRHL3 binding on enhancer and super enhancer activity and function is not known.

overall_design: ChIP-Seq with antibodies to H3K27ac, H3K4me3, and H3K4me1, and an Input control were performed on 24 hour differentiated primary epidermal keratinocytes (NHEK) after siRNA knockdown of GRHL3 (or scramble control). Duplicates of each ChIP were sequenced on Illumina Hi-Seq 2000.

first: Rachel

last: Herndon Klein


title: DNA Methylation Divergence and Tissue Specialization in the Developing Mouse Placenta

city: Los Angeles

first: Benjamin

middle: E

last: Decato

address: 1050 Childs Way

department: Molecular and Computational Biology

institute: University of Southern California

type: Methylation profiling by high throughput sequencing

summary: The placental epigenome plays a vital role in regulating mammalian growth and development. Aberrations in placental DNA methylation are linked to several disease states, including intrauterine growth restriction and preeclampsia. Studying the evolution and development of the placental epigenome is critical to understanding the origin and progression of such diseases. Although high resolution studies have found substantial variation between placental methylomes of different species, the nature of methylome variation has yet to be characterized within any individual species. We conducted a study of placental DNA methylation at high resolution in multiple strains and closely related species of house mice (Mus musculus musculus, Mus m. domesticus, and M. spretus), across developmental timepoints (embryonic days 15 to 18), and between the labyrinthine transport and junctional endocrine layers. We observed substantial genome-wide methylation heterogeneity in mouse placenta compared to other differentiated tissues. Species-specific methylation profiles were concentrated in retrotransposon subfamilies, specifically RLTR10 and RLTR20 subfamilies. Regulatory regions such as gene promoters and CpG islands displayed cross-species conservation, but showed strong differences between layers and developmental timepoints. Partially methylated domains exist in the mouse placenta and widen during development. Taken together, our results characterize the mouse placental methylome as a highly heterogeneous and deregulated landscape globally, intermixed with actively regulated promoter and retrotransposon sequences.

sample_organism:
Mus musculus domesticus
Mus spretus
Mus musculus musculus

overall_design: Two datasets were produced for this study, an inter-specific dataset and intra-specific dataset.  Inter-specific samples are whole placental samples, originated from three species, and are composed of reciprocal hybrid crosses between strains in each species.  Intra-specific samples are purified methylomes of the junctional and labyrinthine zones with equal numbers of samples from male and female mice and embryonic days 15 and 18.

first: Andrew

middle: D

last: Smith

first: Matthew

last: Dean


title: Bi-directional regulation between circadian rhythm and neurodegeneration-associated FUS

city: ShangHai

first: Haifang

last: Wang

address: NO 320, Yueyang Road

department: functional genomics group

institute: PICB

summary: Circadian rhythms are daily physiological and behavioral changes governed by an internal molecular clock, and dysfunctions in circadian rhythms have long been associated with various neurodegenerative diseases. Abnormal sleep-wake cycle often precedes the onset of cognitive and motor symptoms in patients, while the pathological changes may further exacerbate the disturbance in circadian cycle. It is unclear whether dysregulated circadian rhythm is a consequence of, or a contributing factor for, neurodegeneration. In addition, the evidence directly connecting the neurodegeneration-associated proteins to core circadian clock gene expression remains sparse. Here we show that FUS, a RNA-binding protein implicated in the pathogenesis of ALS and frontotemporal dementia, exhibits a bi-directional regulation with circadian rhythm. Our meta-analysis of RNAseq datasets and subsequent biochemical analysis revealed FUS as a gene regulated by circadian oscillation. Furthermore, NR1D1 binds the FUS promoter and regulates the amplitude of FUS oscillation. Meanwhile, FUS is recruited by transcriptional co-repressor PSF, and is found in the same complex as Bmal-Clock  to repress Per2 expression. More strikingly, in cells and brain tissues from homozygous knock-in rats, the pathogenic R521C mutant FUS significantly alters the oscillation patterns of core circadian genes even at young age. Therefore, our results have revealed a novel bi-directional mechanism whereby dysregulated circadian clock and FUS expression may exacerbate neurodegeneration via mutual influence.

overall_design: Cortex, liver, spinal cord profiles of 3.3-month old WT and R521C knock in rat were collected at CT0, CT4, CT8, CT12, CT16, CT20 and  generated by deep sequencing using Illumina HiSeq 2000.

first: Xin

last: Jiang

first: Tao

last: Zhang

first: Jin

last: Xu


title: Deciphering the Regulatory Network between the SREBP pathway and Protein Secretion in Neurospora crassa

status: Public on Apr 11 2017

first: Lina

last: Qin

address: 2151 Berkeley way

institute: University of Carlifornia

summary: Sterol Regulatory Element Binding Proteins (SREBPs) are conserved from yeast to mammalian cells and function in regulating sterol homeostasis. In fungi, the SREBP pathway has been implicated in the adaptation to hypoxia and in virulence. In Neurospora crassa and Trichoderma reesei, the SREBP pathway also negatively regulates protein secretion under lignocellulolytic conditions. Here we utilized global transcriptional profiling combined with genetic and physiological analyses to address the regulatory link between the SREBP pathway and protein secretion in N. crassa. Our results demonstrated that the function of the SREBP pathway in ergosterol biosynthesis and adaptation to hypoxia was conserved in N. crassa. Under lignocellulolytic conditions, the SREBP pathway was highly activated, resulting in the reduced expression of lytic polysaccharide monooxygenases, which require molecular oxygen for catalytic activity. Additionally, activation of the SREBP pathway under lignocellulolytic conditions repressed a set of genes predicted to be involved in the ER stress response. Here we show that the inability of a hac-1 mutant, which bears a deletion of the major regulator of the unfolded protein response (UPR), to efficiently produce cellulases and utilize cellulose was suppressed by mutations in the SREBP pathway. The analyses presented here demonstrated new functions of SREBP pathway, including linkages to the UPR and provide new clues for genetic engineering of filamentous fungi to improve their production of extracellular proteins.

overall_design: Examination of mRNA levels of four strains WT, Δsah-2, Mclr-2 and Mclr-2Δsah-2 strains grown in sucrose minimal media for 24 hours after a shift from 16 hours in VMM by RNA -Seq. Each sample was set as three biological replicates.

first: Vincent

last: Wu

first: N

last: Glass