title: Distinct gene regulatory programs define the inhibitory effects of LXRs and PPARG on cancer cell proliferation

city: Memphis

first: Daniel

last: Savic

state: Tennessee

address: 262 Danny Thomas Place

department: Pharmaceutical Sciences

institute: St. Jude Children's Research Hospital

summary: The liver x receptor (LXR) and peroxisome proliferator-activated receptor gamma (PPARG) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis that impact tumor cell metabolism and proliferation. To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics and nuclear receptor binding patterns. By integrating these orthogonal datasets over time, we uncovered the regulatory architecture of LXR and PPARG activity. Both sets of nuclear receptors inhibited cell proliferation and increased cellular oxidative stress, validating results from independent studies. Despite this metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions altering to repressive gene regulatory activities at late time points through RNA polymerase II (RNAP2) promoter pausing. This exhaustive dataset illustrates the complexity of genome function and structure by elucidating how common phenotypic outcomes are genetically encoded through diverse transcriptional programs. Our results further provide a detailed, molecular framework that can be applied to better understanding how extracellular nutrients impact cancer cell physiology and highlight the importance of study designs that incorporate complementary genomic features in a dynamic manner.

overall_design: ChIP-seq and RNA-seq experiments were performed in HT29 cells using vehicle control (DMSO) or after drug treatment (GW3965 and rosiglitazone).

first: R

last: Myers


title: Cas9 target DNA specificity in vitro and in vivo (S. cerevisiae)

status: Public on Jun 10 2016

city: Stanford

first: Andrew

last: Fire

state: California

address: 300 Pasteur Drive Lane building L302

department: Genetics

country: USA

institute: Stanford

summary: Cas9, a CRISPR RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although Cas9 offers remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of Cas9 target::homology requirements, we compared mismatch tolerance for a specific Cas9::gRNA complex in vitro and in vivo (in Saccharomyces cerevisiae). A variety of truncated and full-length gRNAs (with 17, 18, and 20 nucleotides of complementarity sequence) were used. In each case, we observed notable differences between in vitro and in vivo Cas9 cleavage specificity profiles, with a more stringent effect of mismatches on activity seen in vivo. Increased specificity of the 18 nt complementarity truncated gRNA was evident in vivo, but not in vitro. Overall, this study highlights differences in the specificity of Cas9 cleavage between controlled in vitro conditions and complex and chromatinized in vivo conditions.

overall_design:
We adapted a previous high throughput sequencing approach (doi: 10.1093/nar/gku1102) to assess the effects of single base variants in vivo. We used a polymorphic random variant library matched to a specific trigger sequence (for which we used a segment from the C. elegans unc-22 gene, previously designated ‘unc-22A’ (doi: 10.1093/nar/gku1102). Cas9 in vivo and in vitro assays were carried out with four different gRNAs incorporating sequence from the unc-22A trigger segment.  The four segments incorporate 17 nt, 18 nt, 20 nt, and 20+G nt of unc-22A complementarity respectively.
The retention is calculated based on PMID: 25399416.
File names denote the experiment-> gRNAname_gRNAlength_(invitro or invivo)_(incubation or induction time)_IlluminaRunID.dat
The files contain all normalized sequences in column one with their calculated retentions in column 2.

first: Becky

last: Xu

first: Hua

last: Fu


title: FUS KO mRNA sequencing and anti-FUS RNA immunoprecipitation sequencing

status: Public on Jun 13 2016

city: Bern

first: Oliver

last: Muehlemann

address: Freiestrasse 6

country: Switzerland

institute: University of Bern

type: Expression profiling by high throughput sequencing

summary:
The aim of our study is to identify the role of FUS in shaping the transcriptome. RNA-seq of two FUS KO clones was performed and compared to wt; for each, four replicates were sequenced.
RNA molecules associated with the FUS protein were determined by means of a RNA immuno-precipitation, followed by high-throughput sequencing. Total RNA was used as a control.
SH-SY5Y cells were used for both experiments.

overall_design:
RNA-seq: 4 wt samples, 4 A4 KO samples, 4 A5 KO samples.
RIP-seq: 1 input control sample, 3 anti-FUS IP replicates.

first: Marc

middle: D

last: Ruepp

first: Martino

last: Colombo


title: Growth-phase dependent regulation of transcription by Rsd and 6S RNA

city: Bangalore

first: Avantika

last: Lal

address: GKVK, Bellary Road

country: India

institute: National Centre for Biological Sciences

summary: This entry refers to transcriptome analysis of five E. coli strains: E. coli K-12 MG1655, single deletions of rsd, ssrS, and rpoS, as well as a double deletion of rsd and ssrS, in five growth phases.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: 25 samples (5 strains in 5 growth phases) with 2 replicates each.

first: Aswin

last: Seshasayee


title: Sigma38 binding sites in E. Coli

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ChIP-seq to identify sigma38 binding sites in wild-type and delta ssrS (6S RNA knockout) strains of E. Coli K-12 MG1655, during stationary phase

platform_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: ChIP-seq using antibody against sigma38 in wild-type and ssrS deletion strain. Two replicates for wild type and one replicate for ssrS deletion.


title: An ectopic network of transcription factors regulated by Hippo signaling drives growth and invasion of a malignant tumor model

city: Marseille

first: Delphine

last: Potier

address: 27 boulevard Lei Roure

country: France

institute: CRCM

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

overall_design: Refer to individual Series


title: An ectopic network of transcription factors regulated by Hippo signaling drives growth and invasion of a malignant tumor model [larval wild type discs]

summary: Cancer cells have abnormal gene expression profiles, however, the transcription factors and the architecture of the regulatory network that drive cancer specific gene expression is often not known. Here we studied a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with high-throughput sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, we discovered that the bulk of the tumor specific gene expression is driven by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/AIB1, and Mef2. Notably, many of these transcription factors are also hyperactivated in human tumors. Bioinformatics analysis predicted that these factors directly regulate the majority of the tumor specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness and knock-down of individual factors caused a reversion of the tumor specific expression profile, but had no observable effect on normal tissues. We further found that the Hippo pathway effector Yki/Sd was strongly activated in tumor cells and initiated cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identified an ectopic yet highly ordered network of master regulators that control tumor cell specific gene expression.

overall_design: RNA-seq gene expression profiling across Drosophila 3rd instar larval wild type wing discs and genetic perturbations of wts.

first: Mardelle

last: Atkins

first: Lucia

last: Romanelli

first: Jelle

last: Jacobs

first: Jana

last: Mach

first: Fisun

last: Hamaratoglu

first: Stein

last: Aerts

first: Georg

last: Halder


title: CX3CR1/Fractalkine receptor expression separates memory CD8+ T cells with distinct functional profiles (RNA-seq)

city: Bonn

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

country: Germany

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

summary: Memory T cells are important for protective immunity against infectious microorganisms. Such protection is achieved by cooperative action of memory T cell populations that differ in their tissue localization and functionality. We report on the identification of the fractalkine receptor CX3CR1 as marker for stratification of memory T cells with cytotoxic effector function from those with proliferative function in both, mice and man. Based on CX3CR1 and CD62L expression levels four distinct memory T cell populations can be distinguished based on their functional properties. Transcriptome and proteome profiling revealed that CX3CR1 expression was superior to CD62L to resolve memory T cell functionality and allowed determination of a core signature of memory T cells with cytotoxic effector function. This identifies a CD62Lhi CX3CR1+ memory T cell population with an identical gene signature to CD62LlowCX3CR1+ effector memory T cells. In lymph nodes, this so far unrecognized CD62LhiCX3CR1+ T cell population shows a distinct migration pattern and anatomic positioning compared to CD62LhiCX3CR1neg TCM. Furthermore, CX3CR1+ memory T cells were scarce or absent during chronic HBV, HCV and HIV infection in man and chronic LCMV infection in mice confirming the value of CX3CR1+ in understanding principles of protective immune memory.

overall_design: CD8+ T cells were isolated and directly assessed. After harvesting, cells were immediately lysed in Trizol (Invitrogen) before storage at -80°C for RNA isolation.

first: J

middle: P

last: Böttcher

first: M

last: Beyer

last: Sander

middle: L

first: P

middle: A

last: Knolle


title: Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor

city: Portland

first: Joshi

last: Alumkal

state: OR

address: 3303 SW Bond Ave. CH14R

institute: Knight Cancer Institute

summary: Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation.

overall_design: RNA-seq profiles of prostate cancer cell lines to understand gene expression associated with enzalutamide treatment

first: Daniel

middle: J

last: Coleman

first: Kathryn

last: Van Hook

first: Carly

last: King

first: Jacob

last: Schwartzman

first: Robert

last: Lisac

first: Joshua

last: Urrutia

first: Archana

last: Sehrawat

first: Josha

last: Woodward

first: Nicholas

last: Wang

first: Roman

last: Gulati

first: George

middle: V

last: Thomas

first: Tomasz

middle: M

last: Beer

first: Martin

last: Gleave

first: James

middle: E

last: Korkola

first: Lina

last: Gao

first: Laura

last: Heiser


title: The regulatory role of ferric uptake regulator (Fur) during anaerobic respiration of Shewanella piezotolerans WP3

city: Shanghai

first: Xinwei

last: Yang

address: #800, Dongchuan Road

country: China

institute: SJTU

summary: Purpose:  The goal is to study the regulatory role of ferric uptake regulator (Fur) during anaerobic respiration in Shewanella piezotolerans WP3

overall_design: mRNA profiles of WP3 wild type (WP3) and the fur mutant (Fur) were generated by deep sequencing using Illumina HiSeq 2000.


title: Altered expression of genes in AP4 knockdown and overexpression

status: Public on Jun 09 2016

city: Guangzhou

first: Chen

last: Changhao

address: Department of Urology, Sun Yat-sen Memorial Hospital, 107, W. Yanjiang Road, Yuexiu District, Guangzhou,Guangdong Province, PR China

institute: Sun Yat-sen Memorial Hospital

summary:
The transcription factor AP4/TFAP4/AP-4 is a ubiquitously expressed basic helix-loop-helix leucine-zipper (bHLH-LZ) transcription factor, which forms homodimer that binds to the consensus E-box motif 5-CAGCTG-3. Prostatic adenocarcinoma (PAC), also known as prostate cancer, is the second most common malignancy of men with 914,000 new cases and approximately 258,000 deaths worldwide in 2008. The goal of this study is to identify the target genes of AP4 in prostate cancer. Our results demonstrated that the genes regulated by AP4 are involved in a variety of biological functions, such as proliferation and invasion.
We used microarrays to detail the different expression of genes in AP4 knockdown and overexpression and identified distinct classes of up-regulated genes during this process.

overall_design: Prostate cancer cell line PC-3 was transfected with the control siRNA or AP4 siRNA for 48h. In addition, we applied with AP4, AP4△bHLH adenovirus and empty vector virus transfected PC-3 cell. The RNA of the transfected cells was extracted and hybridized on Affymetrix microarrays. We identified the putative target genes of AP4 in both knockdown and overexpression by comparing the different expressed genes among si-NC vs. si-AP4; AP4 vs. AP4△bHLH vs. vector. We further validated the data of microarray in PC-3 and LNCaP by qPCR and Western blotting.

first: Changhao

last: Chen

first: Qiuhui

last: Pan

first: Feng

last: Su

first: Xu

first: Wang

last: He

first: Jian

last: Huang

first: Tianxin

last: Lin


title: Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network

city: Boston

first: Caroline

middle: Attardo

last: Genco

state: MA

address: 136 Harrison Ave

institute: Tufts University

summary: In this study wild-type, fur mutant, and complemented fur mutant strains of the human pathogen Neisseria gonorrhoeae F62 were grown under high (100 uM iron) or low (100 uM desferal) iron conditions to identify genes whose expression was regulated by iron and/or Fur

overall_design: Examination of each of three strains under two conditions by RNA-seq

first: Ryan

last: McClure

first: Kathleen

last: Nudel


title: CX3CR1/Fractalkine receptor expression separates memory CD8+ T cells with distinct functional profiles

first: Joachim

last: Schultze

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Lead Modulates trans- and cis-eQTLs in Drosophila melanogaster Heads

city: Detroit

first: Wen

last: Qu

state: MI

address: 275 E. Hancock Ave., Room  291.1

department: Pharmacology

institute: Wayne State University

summary: The goal was to study the effects of lead exposure on gene expression and identify the lead-responsive genes. After detecting 1,536 cis-eQTLs (FDR ≤ 10%) and 952 trans-eQTLs, we focused our analysis on Pb-sensitive “trans-eQTL hotspots”.

overall_design: 158 randomly selected Drosophila Synthetic Population Resource (A2) samples (control 79 samples and Pb-treated) without replicates

first: Douglas

last: Ruden


title: An ectopic network of transcription factors regulated by Hippo signaling drives growth and invasion of a malignant tumor model [larval imaginal discs]

first: Delphine

last: Potier

overall_design: RNA-seq gene expression profiling across Drosophila 3rd instar larval imaginal discs in a control and different genetic perturbations.

first: Mardelle

last: Atkins

first: Lucia

last: Romanelli

first: Jelle

last: Jacobs

first: Jana

last: Mach

first: Fisun

last: Hamaratoglu

first: Stein

last: Aerts

first: Georg

last: Halder


title: RNA-seq identifies genes that are regulated by SAGA deubiquitinase activity in glial nuclei of the Drosophila central nervous system during larval development

city: WEST LAFAYETTE

first: Vikki

last: Weake

state: INDIANA

address: 175 S. University St.

department: Biochemisty

institute: Purdue University

summary:
Purpose: Next-generation sequencing (NGS) was used to elucidate genes in Drosophila visual system glia that are sensitive to impairment of the deubiquitinase activity of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex.
Methods: The nuclear envelope of glial cells was genetically labled with a KASH-GFP fusion protein using the Gal4/UAS binary expression system and repo-Gal4 as driver in wild-type, nonstop and sgf11 third instar larvae. GFP-labeled glial nuclei were released by homogenization and then isolated from the central nervous system/eye antennal disc complex of Drosophila melanogaster third instar larvae from each genotype, and mRNA was extracted. For RNA-seq, dsDNA was generated using the Ovation RNA-Seq System V2 (Nugen technologies), DNA libraries were generated using standard methods (Illumina), and paired-end 100-base fragements were sequenced on an Illumina HiSeq2500 next generation sequencer.
Result: EdgeR analysis with significance cut-off of FDR<0.01 identified 629 genes that are significantly downregulated, and 779 genes that are significantly upregulated, in both the nonstop and sgf11 visual system glia as compared to wild-type glia.

overall_design: The active transcriptome (nuclear mRNA) of glial cells from wild type, nonstop and sgf11 were generated by deep sequencing of four biological replicates for each genotype using Illumina HISeq2500

first: Jingqun

last: Ma


title: Effect of loss of Jmjd1c on normal hematopoiesis

city: New York

first: Noushin

last: Farnoud

state: NY

address: 417 East 68th Street

institute: Memorial Sloan Kettering Cancer Institite

summary: Analysis of gene expression profile of LSK (Lin-Sca-1+ c-Kit+) isolated from Jmjd1c f/f Vav1Cre or Vav1Cre controls. Loss of Jmjd1c minimally affected HSC in homeostatsis while impiars HSC function in response to stree such as transplantation and 5-Fu treatment.  These results provide insight into the role of Jmjd1c in normal hematopoiesis.

overall_design: LSK cells were isolated from bone marrow of seven weeks old  Jmjd1c f/f Vav1Cre or Vav1Cre controls mice by flow acivated cell sorting and  subjected  to RNA-seq.

first: N

last: Zhu

middle: F

last: Rahnamay

first: Amit

last: Sinha

first: Richard

last: Koche

first: S

last: Armstrong


title: Effect of loss of Jmjd1c on MLL-AF9 leukemia

summary: Analysis of gene expression profile of MLL-AF9 leukemia cells 6 days after loss of Jmjd1c. Loss of Jmjd1c induces differentiation and apoptosis in MLL-AF9 leukemia cells. These results provide insight into the role of Jmjd1c in MLL leukemia.

overall_design: We used primary Jmjd1c f/f MLL-AF9-GFP leukemia cells from four leukemic mice and delivered Cre-Tomato by retroviral infection. Cells infected with virus made from empty vector was used as control. We sorted GFP and Tomato double positive cells 6 days after Cre infection and subjected the samples to RNA-seq.


title: Effect of loss of Jmjd1c on H3K9me2 level across the genome

summary: Analysis of H3K9me2 level in MLL-AF9 leukemia cells upon deletion of Jmjd1c. Jmjd1c has been shown to be a H3K9me1/2 demethylase and these results provide insight into its role in MLL-Af9 leukemia

overall_design: We used primary Jmjd1c f/f MLL-AF9-GFP leukemia cells and delivered Cre-Tomato retroviral infection. Cells infected with virus made from empty vector was used as control (MIT).  We sorted GFP and Tomato double positive cells 6 days after infection and subjected the samples to ChIP-seq. We also carried out ChIP-seq in single cell clone (SSC) derived from methylcult culture of Jmjd1c f/f MLL-AF9-GFP/ Cre cells.


title: Impact of a heterotroph, Alteromonas MIT1002, on the transcriptome of Prochlorococcus NATL2A

city: Cambridge

first: Steven

last: Biller

address: 15 Vassar St, MIT 48-424

department: Civil and Environmental Engineering

institute: MIT

summary: We examine how the transcriptome of Prochlorococcus strain NATL2A changes in the presence of a naturally co-occurring heterotroph, Alteromonas macleodii MIT1002. Significant changes in the Prochlorococcus transcriptome were evident within six hours of co-culture, with groups of transcripts changing in different temporal waves. Many transcriptional changes persisted throughout the 48-hour experiment, indicating that the presence of the heterotroph affected a stable shift in Prochlorococcus physiology. These initial transcriptome changes largely correspond to reduced stress conditions within Prochlorococcus, as inferred from decreases in relative abundance for transcripts encoding DNA repair enzymes and many members of the ‘high-light inducible’ family of stress response proteins. Notable changes were also seen in transcripts encoding components of the photosynthetic apparatus (particularly an increase in PSI subunits and chlorophyll synthesis enzymes), ribosomal proteins and biosynthetic enzymes. Changes in secretion-related proteins and transporters also highlight the potential for metabolic exchange between the two strains.

sample_organism:
Alteromonas macleodii
Prochlorococcus marinus str. NATL2A

overall_design: At each of 7 timepoints, samples from 3 biological replicate co-cultures are compared to 3 biological replicate axenic Prochlorococcus cultures that serve as a control.

first: Allison

last: Coe

first: Sallie

middle: W

last: Chisholm


title: Effect of loss of Jmjd1c on Hoxa9/Meis1 leukemia

summary: Analysis of gene expression profile of Hoxa9/Meis1 leukemia cells 6 days after loss of Jmjd1c. Loss of Jmjd1c induces differentiation and Hoxa9/Meis1 leukemia cells. These results provide insight into the role of Jmjd1c in AML with elelvated expression of Hoxa9.

overall_design: We used primary Jmjd1c f/f Hoxa9/Meis1 neo leukemia cells from three leukemic mice and delivered Cre-Tomato by retroviral infection. We sorted Tomato positive cells 6 days after Cre infection and subjected the samples to RNA-seq.


title: Jmjd1c


title: mRNA-seq from Nutlin-3a, doxorubicin, and DMSO treated HCT116 p21-/- cells

city: Jena

first: Martin

last: Fischer

state: Thuringia

address: Beutenbergstraße 11

department: Computational Biology

institute: Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)

summary: We sequenced mRNA from HCT116 p21-/- cells treated with Nutlin-3a, doxorubicin, or DMSO for 24 h.

overall_design: Examination of mRNA levels from HCT116 p21-/- cells treated with Nutlin-3a, doxorubicin, or DMSO for 24 h using four replicates each.

first: James

last: DeCaprio


title: A positive regulatory loop between a Wnt-regulated non-coding RNA and ASCL2 controls intestinal stem cell fate

city: Vari

first: Pantelis

last: Hatzis

state: Attiki

address: 34 Fleming Str

department: Molecular Biology and Genetics

country: Greece

institute: Biomedical Sciences Research Center 'Alexander Fleming'

summary: The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/β-catenin transcriptional complex is the primary transforming factor in colorectal cancer. Despite significant recent inroads, the full complement of Wnt target genes and the mechanisms of regulation remain incompletely understood. Here we identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its close neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition/physical contact of its own promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 expression. This feedforward regulatory loop controls stem cell-related gene expression and is highly amplified in colorectal cancer.

overall_design: Derivatives of Ls174T colon cancer cells, overexpressing the Tet repressor were used for the construction of inducible overexpressing a shRNA against the WiNTRLINC1 long non coding RNA upon treatment with doxyxycline. siRNAs against WiNTRLINC1 were designed with the siDesign center tool from Dharmacon and their sequences were used for the construction of the shRNA stem loop structure as described in EMBO Rep. 2003 Jun;4(6):609-15. The modified pTER vector was used as a backbone for constructing the shRNA cassette as described in EMBO Rep. 2003 Jun;4(6):609-15. Positive cell clones were screened with RT-PCR in order to validate the efficiency of the knockdown of WiNTRLINC1. The Ls174T derivative cell line inducibly overexpressing a shRNA against ASCL2 has been described previously  in Cell. 2009 Mar 6;136(5):903-12. RNA deep sequencing was performed in the WiNTRLINC1 KD and ASCL2 KD cells compared to controls cells in order to detect changes in gene expression due to the loss of either WiNTRLINC1 or ASCL2.

first: Antonis

last: Giakountis

first: Panagiotis

last: Moulos

first: Vasiliki

last: Zarkou

first: Christina

last: Oikonomou

first: Vaggelis

last: Harokopos

first: Artemis

middle: G

last: Hatzigeorgiou

last: Reczko


title: Pdx1-Oc1 cooperatively drive the induction of the endocrine pancreatic program

city: Philadelphia

first: Diana

middle: Elena

last: Stanescu

state: PA

address: 3400 Civic Center Blvd

department: IDOM

institute: University of Pennsylvania

summary: We report the impact of heterozygous loss of either Pdx1 or Oc1 on the developing pancreas at e15.5

overall_design: mRNA of mouse pancreata at embryonic day 15.5 from control, Pdx1Lac/+, Oc1+/- and double heterozygous (Pdx1LacZ/+;Oc1+/-) embryos

first: Doris

last: Stoffers


title: Genome-wide maps of chromatin binding site for Mdm2.

city: Montpellier

first: Laetitia

middle: Karine

last: Linares

address: 208 rue des apothicaires

institute: INSERM

summary: The MDM2 oncoprotein is an essential component of the p53 pathway. Although MDM2 has been widely described as a major negative regulator of the p53 tumor suppressor, growing evidences support the notion that Mdm2 functions are also mediated through p53 independent mechanisms. We found that MDM2 is recruited on chromatin. By ChIP-Seq (using MDM2 antibody) of H1299 cells exhibiting MDM2 recruitment on chromatin, we identified at the whole genome level MDM2 binding sites. Both input and IPped-DNA were exhaustively sequenced and mapped on human genome

overall_design: Genome wide mapping ofMdm2 binding in H1299

first: Romain

last: Riscal

first: Laurent

last: Le Cam

middle: K


title: Identification of symmetric methyltion of histone H4 arginine 3 on KSHV genome

status: Public on Jun 08 2016

city: Reno

first: Subhash

middle: C

last: Verma

state: NV

address: 1664 N Virginia Street, MS 320

department: Microbiology and Immunology

institute: University of Nevada, Reno

summary: The objective of this study was to identify the symmetric methylation sites on KSHV genome during latency and lytic reactivation

overall_design: KSHV infected, TRExBCBL1-RTA cells were induced with doxycycline for 24h to trigger the lytic reactivation, cells were crosslinked and subjected for chromatin isolation and sonication. ChIP assay was performed using anti-H4R3sym antibody


title: Systems level analyses establish CodY as a multifaceted metabolic regulator integrating metabolism, motility and virulence in Listeria monocytogenes

city: Tel Aviv

first: Lior

last: Lobel

state: Israel

address: Ramat Aviv

department: Molecular Microbiology

country: Israel

institute: Tel Aviv University

summary: To gain knowledge on L. monocytogenes differential CodY activity, we set on analyzing CodY’s regulon in L. monocytogenes in both rich and minimal growth conditions using genome-wide sequencing techniques. Remarkably, we identified for the first time a global regulatory role for CodY when BCAAs are limited, that are similar to those within the mammalian niche. Furthermore, our data establish CodY as a central regulator that integrates metabolism, motility, stress responses and virulence in L. monocytogenes.

overall_design:
Examination of CodY regulon in rich and minimal conditions.
Sample codY_LBMM_3 did not correlate with the other two replicates and was therefore omitted.

first: Anat

last: Herskovits


title: TLR2/1 ligand and IFN-g inducible genes in human monocyte-derived macrophages (MDMs)

city: Los Angeles

first: Susan

last: Realegeno

state: CA

address: 615 Charles E Young Dr. South

department: Microbiology, Immunology, & Molecular Genetics

institute: University of California, Los Angeles

summary: Transcriptome profiles for innate and adaptive immune stimuli important for host response against mycobacteria.  Human monocyte-derived macrophages were stimulated with TLR2/1 ligand and interferon-g, stimuli present during innate and adaptive immune responses, respectively.

overall_design: Human monocyte-dervided macrophages from five healthy donors were stimulated with TLR2/1L, IFN-g, or media control for 2, 6, and 24 hours.  RNA-sequencing was performed on a total of 45 samples.

first: Robert

last: Modlin

first: Matteo

last: Pellegrini


title: Nucleation of DNA Repair Factors by FOXA1 Links DNA Demethylation to Transcriptional Pioneering

city: Beijing

first: Yu

last: Zhang

address: Xueyuan Road

institute: Peking University Health Science Center

type: Methylation profiling by high throughput sequencing

summary: We report that the winged helix transcription factor FOXA1 is unexpectedly associated with components of single and double stranded-DNA repair complexes. Biochemical studies and high-throughput approaches validated the hierarchical composition of this FOXA1-nucleated machinery and revealed the dependency on FOXA1 for global targeting of the key repair polymerase POLB. Genome-wide DNA methylomes at single-base resolution demonstrated that FOXA1-DNA repair complex is functionally linked to DNA demethylation in a lineage specific fashion. Loss-of-function studies indicate that a significant portion of FOXA1-bound regions display localized reestablishment of methylation and that the subsets with most consistent hypermethylation are represented by active promoters and enhancers that also exhibit the greatest depletion of POLB following FOXA1 removal. Consistently, forced expression of FOXA1 commits its binding sites to an active DNA demethylation in a POLB dependent manner. Finally, we showed that FOXA1-associated DNA demethylation is tightly coupled with genomic targeting of estrogen receptor and estrogen responsiveness. Together, our results link FOXA1-associated DNA demethylation to its transcriptional pioneering.

overall_design: Genome-wide FOXA1 and POLB binding were examined through ChIP-seq in MCF-7 cells. Whole-genome bisulfite sequencing was performed in replicates in siNS- or siFOXA1-transfected MCF-7 cells.

first: Yongfeng

last: Shang


title: alphaT-catenin in restricted brain cell types and its potential connection to autism

city: Chicago

first: Cara

last: Gottardi

address: 240 East Huron St., McGaw Pavilion, M-323

department: Departments of Medicine

institute: Northwestern University

summary:
RNA-seq analysis was performed between WT and alphaT-cat KO mouse cerebella aiming to discover gene transcripts altered by the loss of alphaT-cat
These altered gene transcripts could be associated with several neurologic disease-relevant pathways

overall_design: Total RNA extracted of cerebellar tissue (n=3) from the brains of WT ad alphaT-cat KO mice

first: Stephen

middle: S

last: Folmsbee

first: Douglas

middle: R

last: Wilcox

first: Koen

last: Tyberghein

first: Pieter

last: De Bleser

first: Warren

last: Tourtellotte

first: Jolanda

last: van Hengel

first: Frans

last: van Roy


title: NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins.  In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target.  Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions.  In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety.  We have functionalized the steric moiety with alkyne for use in click chemistry reactions.  This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques.  We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3.  We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”.  We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment.  In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively.  Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing.  We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown.  PARP-1 depletion caused an increase in Pol II pausing genome-wide.  Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation.

overall_design: Using ChIP-seq (knockdown of Luciferase [control] with or without CDK inhibitor [DRB] treatment, knockdown of PARP-1, knockdown of NELF-E, and re-expression of GFP [control], wild-type NELF-E, mutant NELF-E, or viral NELF inhibitor HDAg-S) in MCF-7 human breast cancer cells and using ChIP-seq (PARP-1 and click-ChIP-seq) in mouse embryonic fibroblasts (MEFs)

first: Bryan

last: Gibson

first: W


title: A TGFbeta-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

city: Bronx

first: David

last: Shechter

state: New York

address: 1300 Morris Park Ave

department: Biochemistry

institute: Albert Einstein College of Medicine

summary: We sequenced mRNA from 3 biological replicates each of A549 lung adenocarcinoma cell lines expressing shRNA against GFP (control), PRMT5, or MEP50. We then determined differential gene expression.

overall_design: Transcriptome analysis of mRNA testing the role of PRMT5 and MEP50 by knockdown in A549 human lung adenocarcinoma cells

first: H

last: Chen

first: D


title: cis-Regulatory analysis of Ptch1

first: Kevin

last: Peterson

address: 1425 San Pablo St.

department: Department of Stem Cell Biology and Regenerative Medicine

institute: Eli and Edythe Broad-CIRM Center for Regenerative Medicine, University of Southern California Keck School of Medicine

summary: Ptch1 is the primary receptor for Hedgehog ligands and functions as a negative regulator of the pathway. Ptch1 is upregulated in response to pathway activation and is a direct target of the Gli family of transcription factors that act as the downstream effectors of the pathwat. The goal of this study was to identify tissue specific Gli2 inputs to Ptch1 during development. Additionally, we assessed the prevalence of the repressive chromatin mark (H3K27me3) during directed differentiated of embryonic stem cells into neural progenitors.

overall_design: Embryonic tissues were microdissected and pooled prior to formaldehyde fixation. ES cell derived neural progenitors were generated using embryoid body differentiation according to standard protocols.

first: Andrew

last: McMahon


title: Comparative analysis of cotton small RNAs in response to salt stress

city: Anyang

first: Zujun

last: Yin

state: Henan

address: 38 Huanghe Dadao, Anyang 455000, Henan, China

department: Cotton Research Institute

institute: Chinese Academy of Agricultural Sciences

type: Non-coding RNA profiling by high throughput sequencing

summary: Plants respond to stress by using multiple gene regulatory mechanisms including the post-transcriptional regulation of gene expression. The stresses suffered by plants under salinity include osmotic stress and ion stress. In this study, three cotton small RNA libraries were constructed and sequenced under normal consideration, osmotic and ionic stress. The length distribution of obtained small RNAs was significantly different between libraries. A total of 228 cotton miRNAs were identified. Of them 24 were novel miRNAs. There were 88 and 75 miRNAs with different expression in response to the influence of osmotic and ionic factors of saline stress, respectively. The identification of these small RNAs as well as elucidating their functional significance broadens our understanding of the post-transcriptional gene regulations in response to salt stress.

overall_design: Three separate cDNA libraries of small RNAs were constructed using total RNA obtained from control samples (CK), the 4 h and 5 d samples. A total of 57,370,554 sequence reads were obtained from three libraries. After removing the 3p and 5p adapter sequences and filtering out low quality "n" sequences, 17,026,070, 15,731,477 and 22,590,391 clean reads remained in the CK, 4 h and 5 d samples, respectively.


title: RNA polymerase II is trapped in initiation states on non-coding RNA transcripts

city: Edinburgh

first: Grzegorz

last: Kudla

address: Crewe Road

department: MRC HGU

country: United Kingdom

institute: University of Edinburgh

summary: The interaction between RNA polymerase II (RNAPII) and RNA processing and packaging factors is strongly influenced by the C-terminal domain (CTD), which consists of multiple heptad repeats that can be differentially phosphorylated at five positions. Here we report strand-specific, high-resolution profiling of the five types of RNAPII CTD phosphorylation in yeast using crosslinking and analysis of modified polymerase (CLAMP). The 5’ regions of protein coding genes showed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P and Ser7P. CTD phosphorylation pattern boundaries were associated with known sites of RNAPII pausing, splicing, and nucleosome positioning. To integrate the distribution of the RNAPII modifications across all transcription units, we developed an eight-state, strand-specific Hidden Markov Model. This identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the Transcription Start Site (TSS) of mRNAs and ncRNAs, and persisted throughout the 1st exon of intron-containing genes. Notably, unstable ncRNAs failed to transition into the elongation states seen on protein coding genes, and their early termination and rapid degradation probably reflect this failure.

overall_design:
19 samples were analyzed in total:
12 samples (1 to 12) from a strain carrying HTP-tagged Rpb1. Duplicate experiments were carried out using antibodies against the different phosphorylation states of S. cerevisiae Rpb1 carboxy-terminal domain. Ser5-P, Ser7-P, Ser2-P, Tyr1-P, Thr4-P as well as a no antibody control.
7 samples (13 to 19) from strains carrying HTP-tagged protein of interest, either Air2, Trf4, Rrp44 or Rrp6 (no antibody were used for these 9 samples).

first: Laura

last: Milligan

first: Van-Anh

last: Huynh-Thu

first: Clementine

last: Delan-Forino

first: Alex

last: Tuck

first: Elisabeth

last: Petfalski

first: Rodrigo

last: Lombraña Pascual

first: Guido

last: Sanguinetti

last: Tollervey


title: Joint-specific DNA transcriptome signatures in rheumatoid arthritis [RNA-seq]

city: La Jolla

first: Gary

last: Firestein

address: 9500 Gilman Dr

institute: UCSD

summary: Stratifying patients on the basis of molecular signatures could facilitate development of therapeutics that target pathways specific to a particular disease or tissue location. Previous studies suggest that pathogenesis of rheumatoid arthritis (RA) is similar in all affected joints. Here we show that distinct DNA methylation and transcriptome signatures not only discriminate RA fibroblast-like synoviocytes (FLS) from osteoarthritis FLS, but also distinguish RA FLS isolated from knees and hips. Using genome-wide methods, we show differences between RA knee and hip FLS in the methylation of genes encoding biological pathways, such as IL-6 signaling via JAK-STAT pathway. Furthermore, differentially expressed genes are identified between knee and hip FLS using RNA-seq. Double-evidenced genes that are both differentially methylated and expressed include multiple HOX genes.  Joint-specific DNA signatures suggest that RA disease mechanisms might vary from joint to joint, thus potentially explaining some of the diversity of drug responses in RA patients.

overall_design: Total RNA-seq from knee and hip joints in rheumatoid arthritis (RA)

first: Rizi

last: Ai

first: Alice

last: Walsh

first: Deepa

last: Hammaker

last: Boyle

first: Wei

last: Wang


title: RNA helicase Spn-E is required to maintain Aub and AGO3 protein levels for piRNA silencing in the germline of Drosophila

city: Moscow

first: Sergei

last: Ryazansky

address: Kurchatov sq., 2

department: Department of Molecular Genetics of Cell

country: Russia

institute: Institute of Molecular Genetics

summary: Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.

overall_design: The fractions of small RNAs (19-29 nt) from testis of Drosophila melanogaster spnE/+ spnE/- strains were sequenced using Illumina HiSeq 2000.

first: Alexei

last: Kotov

first: Mikhail

last: Kibanov

first: Natalia

last: Akulenko

first: Alina

last: Korbut

first: Sergey

last: Lavrov

first: Vladimir

last: Gvozdev

first: Ludmila

last: Olenina


title: Dissecting the direct reprogramming path of fibroblasts into neurons by single cell RNA-sequencing

city: Stanford CA

first: Barbara

last: Treutlein

address: 318 Campus Drive

department: Bioengineering

institute: Stanford University

summary: Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediates through which individual cells progress are largely undefined. Here we used single cell RNA-seq at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity rather than time we reconstructed a continuous reprogramming path. We find that overexpression of a single factor (Ascl1) results in a well-defined initialization causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. However, overexpression of Ascl1 alone leads to abundant alternative fates that are suppressed by the combination of additional factors (Myt1l, Pou3f2). We find transgene silencing and emergence of alternative fates are the major efficiency limits of direct reprogramming. These data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

overall_design: 405 single-cell transcriptomes from 5 time points (0 days, 2 days, 5 days, 20 days and 22 days upon Ascl1 induction) during reprogramming of MEFs into iN cells were analyzed in total; All single cells contain 92 external RNA spike-ins; The following single cell transcriptomes were sequenced: 73 single MEF cells (day 0, iN2), 81 single cells at day 2 (iN1), 55 single cells at day 5 (iN3), 33 single cells at day 20 (iN6) and 73 single cells at day 22 (iN7,iN8) upon Ascl1 induction from virally transfected MEFs. Additionally, we generated the transcriptome of 47 single cells that were induced with Ascl1 for 2 days from a clonal MEF line with a uniform Ascl1 transgene copy number (iN5). Finally, we sequenced the transcriptome of 43 single cells at day 22 (iN4) upon induction of Ascl1, Brn2 and Myt1l. All single cell samples were processed on the microfluidic Fluidigm C1 platform.

first: Stephen

last: Quake


title: T-bet recruits P-TEFb to super-enhancers to regulate T helper cell differentiation (RNA-Seq)

city: London

first: Richard

last: Jenner

address: 72 Huntley Street

department: Cancer Biology

institute: UCL Cancer Institute

summary: The transcription factor T-bet induces differentiation of CD4+ T cells into the Th1 lineage and also allows for a degree of functional plasticity. Here, we show that T-bet acts through super-enhancers to recruit the elongation factor P-TEFb. Th1-specific genes are poised for activation in Th2 cells and P-TEFb recruitment activates transcriptional elongation. T-bet also induces extensive P-TEFb binding at super-enhancers, where it acts to stimulate enhancer RNA transcription. P-TEFb inhibition selectively blocks activation of lineage-specific genes and reverses Th1-associated retinitis pathology. T-bet-mediated recruitment of P-TEFb to super-enhancers at otherwise poised genes provides a model for how lineage-specifying factors promote differentiation towards specific cell fates whilst maintaining a degree of functional plasticity.

overall_design: Strand-specific total and poly-A+ RNA-Seq in Th1 and Th2 cells from two independent donors

first: Hertweck

last: Arnulf


title: MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

status: Public on Jun 07 2016

city: Odense M

first: Susanne

last: Mandrup

address: Campusvej 55

department: Biochemistry and Molecular Biology

country: Denmark

institute: University of Southern Denmark

summary: In this setup we have used RNA-seq to define which cAMP-induced genes are dependent on activity of the JAK kinases or MDM2. Also, we mapped binding of the coactivator CRTC2 to DNA upon forskolin stimulation.

overall_design: Global gene expression analysis in 3T3-L1 preadipocytes as well as p53-/- and p53-/-;mdm2-/- MEFs treated with JAK inhibitor and/or forskolin. Furthermore, we have performed ChIP on CRTC2 in 3T3-L1 preadipocytes stimulated with forskolin and pretreated with either vehicle or the JAK inhibitor P6.

first: Philip

last: Hallenborg

first: Majken

last: Siersbæk

first: Inigo

last: Barrio-Hernandez

first: Ronni

last: Nielsen

first: Karsten

last: Kristiansen

first: Lars

last: Grøntved

first: Blagoy

last: Blagoev


title: Comparative cistromics reveals genomic crosstalk between FOXA1 and ERα in tamoxifen-associated endometrial carcinomas

city: Amsterdam

first: Marjolein

last: Droog

state: Noord-Holland

address: Plesmanlaan 121

department: Molecular Pathology

country: Netherlands

institute: the Netherlands Cancer Institute

summary: Tamoxifen, which is used to treat breast cancer, increases the risks of endometrial cancer. In this study, we performed a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. Our results define conserved sites for genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer.

overall_design: Binding sites were mapped of Estrogen Receptor alpha (ERa) and FOXA1 in five tamoxifen-associated endometrial carcinoma's (tumor A, tumor B, tumor C tumor D and tumor E). Biological replicated were produced for tumor B.

first: Ekaterina

last: Nevedomskaya

first: Yongsoo

last: Kim

first: Koen

middle: D

last: Flach

first: Wilbert

last: Zwart


title: Comparison of HCC cell lines and primary HCCs-RNAseq data

city: Shanghai

first: Lijian

last: Hui

address: yueyang road 320

department: SIBCB

institute: CEMCS, CAS

summary: There are concerns about whether cancer cell lines could faithfully represent the matched primary cancer cells. Comparison of the HCC cell lines and primary HCCs demonstrated that, during long-term in vitro culture, cell lines retain the genetic landscape of the matched primary HCCs.

overall_design: RNAseq and WGS comparison of of 9 matched primary HCCs, early-passage PDCs and late-passage cell lines.

first: Zhixin

last: Qiu


title: T-bet activates poised Th1 genes through Mediator and the Super Elongation Complex [ChIP-Seq]

first: Richard

last: Jenner

summary: The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through super-enhancers in human and mouse Th1 cells to recruit Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1-specific genes are poised in Th2 cells while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and super-enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, but T-bet- and NF-κB-mediated pathways converge to allow P-TEFb recruitment. These data support a model in which lineage-specifying factors allow recruitment of P-TEFb to poised genes to promote differentiation of alternative T cell fates.

overall_design:
Indentification of P-TEFb binding sites in human Th1 and Th2 cells
Indentification of RelA binding sites in human Th1 cells treated with BAY 11-7082 and DMSO control
Indentification of P-TEFb binding sites in human Th1 cells treated with BAY 11-7082 and DMSO control
Indentification of AFF4 binding sites in human Th1 cells treated with BAY 11-7082 and DMSO control
Indentification of BRD4 binding sites in human Th1 cells treated with BAY 11-7082 and DMSO control
Indentification of MED1 binding sites in human Th1 cells treated with BAY 11-7082 and DMSO control
Indentification of P-TEFb binding sites in mouse wild-type and T-bet -/- Th1 and wild-type Th2 cells
Indentification of RelA binding sites in mouse wild-type and T-bet -/- Th1 cells
Indentification of Brd4 binding sites in mouse wild-type and T-bet -/- Th1 cells
Indentification of Med1 binding sites in mouse wild-type and T-bet -/- Th1 cells
Indentification of Aff4 binding sites in mouse wild-type and T-bet -/- Th1 cells
Indentification of P-TEFb binding sites in EL4-GFP and EL4-T-bet cells
Indentification of Flag-T-bet binding sites in EL4-T-bet-mPlum and EL4-Gata3-GFP cells
Indentification of RelA binding sites in EL4-GFP and EL4-T-bet cells
Indentification of Brd4 binding sites in EL4-GFP and EL4-T-bet cells
Indentification of Med1 binding sites in EL4-GFP and EL4-T-bet cells
Indentification of Aff4 binding sites in EL4-GFP and EL4-T-bet cells
Indentification of RNA polymerase II binding sites in human Th1 and Th2 cells
Mapping of H3K4me3 histone modifications in human Th1 and Th2 cells
Mapping of H3K4me3 histone modifications in human CCR5+ memory Th1
Mapping of H3 histone in human CCR5+ memory Th1
Mapping of H3K27ac histone modifications in human Th1 and Th2 cells
Mapping of H3K4me3 histone modifications in mouse wild-type and T-bet-/- Th1 cells
Mapping of H3 histone in mouse wild-type and T-bet-/- Th1 cells


title: Emergence of neural crest identity is a key early step in melanoma formation [ChIP-Seq]

city: Boston

first: Leonard

last: Zon

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: The “cancerized field” concept posits that cells of a given tissue share a potentially oncogenic mutation or insult and are thus cancer-prone, yet only discreet clones within the field initiate tumor formation.  In melanoma, tumors frequently (~50%) carry the oncogenic BRAFV600E mutation that is also nearly always present in benign nevi that rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors and is then specifically re-expressed in melanoma tumors.  Here, we show by live imaging transgenic zebrafish crestin reporters that single melanocytes in a cancerized field with oncogenic BRAFV600E and p53 tumor suppressor loss undergo a change that recapitulates the neural crest progenitor (NCP) state, and patches of these cells initiate early melanoma. The crestin element is regulated by the NCP transcription factor sox10.  Forced sox10 overexpression in melanocytes accelerated melanoma formation, consistent with reprogramming to the NCP state and activation of super-enhancers that lead to melanoma. Our work highlights the importance of the reemergence of the NCP state as a barrier to melanoma initiation.

overall_design: ChIP-seq analysis of histone 3 lysine 27 acetylation in human A375 melanoma cells treated with DMSO

first: Charles

last: Kaufman

first: Song

last: Yang

first: Yi

last: Zhou

middle: I


title: Transcriptome analysis of HSV-1 infected dorsal root ganglia and large intestinal musculara

city: New Haven

first: William

last: Khoury-Hanold

state: CT

address: The Anlyan Center, 300 Cedar St.

department: Immunobiology

institute: Yale University

summary: Following genital HSV-1 infection in mice, virus spreads to and infects the sensory neurons of the dorsal root ganglia (DRG) and the enteric neurons situated within the large intestinal musculara (LIM). We used RNA sequencing to simultaneously study the host and viral transcriptomes at these two distinct sites of infection. We find that transcription across the HSV-1 genome is weak and incomplete in sensory neurons whereas all known HSV-1 coding genes are robustly expressed in the enteric neurons. Furthermore, a T cell activation signature dominated the immune response to HSV-1 infection in the DRG. In contrast, an innate inflammatory signature was observed in the LIM.

overall_design: Duplicate samples of DRG and LIM from mock or HSV-1 infected mice were collected six days post infection. mRNA transcriptome profiles were generated by deep sequencing using the Illumina 2500 HiSeq platform.

first: Akiko

last: Iwasaki


title: Convergent evolution of peptide-based quorum sensing required for virulence in a eukaryotic pathogen

city: San Francisco

first: Christina

middle: M.

last: Homer

department: Biochemistry & Biophysics

institute: UCSF

summary: Bacterial quorum sensing (QS) systems are characterized by the regulated biogenesis and sensing of specialized signaling molecules. Here, we describe a peptide, Qsp1, secreted by the fungal pathogen Cryptococcus neoformans. Mutants lacking Qsp1 are attenuated for infection, pulmonary accumulation, and growth within macrophages. Qsp1 promotes density-dependent cell wall integrity and resistance to extreme environments. Qsp1 is also required for a secreted aspartyl protease activity required for virulence. Further biochemical and large-scale genetic investigations reveal that cleavage of Qsp1 from the C-terminal of a pro-peptide requires a cell-associated serine protease and Qsp1 sensing requires a predicted oligopeptide importer. Strikingly, these features closely mirror the hallmarks of a peptide-based QS system found in gram-positive bacteria, despite no ancestral relationship between individual components. Our studies thus reveal a convergently evolved, peptide-based QS system required for the virulence of a fungal pathogen.

overall_design: RNA-Seq or ChIP-Seq to compare multiple Cryptococcus neoformans genotypes using the HiSeq 2500 platform

first: Diana

last: Summers

first: Alexi

last: Goranov

first: Starlynn

last: Clarke

first: Darin

middle: L

last: Wiesner

first: Jolene

last: Diedrich

first: James

last: Moresco

first: Dena

last: Toffaletti

first: Rajendra

last: Upadhya

first: Ippolito

last: Caradonna

first: Sarah

last: Petnic

first: Veronica

last: Pessino

middle: A

last: Cuomo

first: Jennifer

last: Lodge

first: John

last: Perfect

last: Yates III

first: Kirsten

last: Nielsen

last: Craik

first: Hiten

last: Madhani


title: Identification of KSHV RTA and K8 binding sites on viral genome

city: Reno

first: Subhash

last: Verma

address: 1664 N Virginia Street, MS 320

department: Microbiology and Immunology

institute: University of Nevada, Reno

summary: The objective of this study was to identify the binding sites of KSHV encoded imemdiate early protein, RTA and early protein, K8 on KSHV genome by chromatin immunoprecipitation assay and sequencing of the DNA bound to RTA and K8

overall_design: KSHV infected, TRExBCBL1-RTA cells were induced with doxycycline for 24h to trigger the lytic reactivation, cells were crosslinked and subjected for chromatin isolation and sonication. ChIP assay was performed using purified antibodies for RTA and K8.


title: Ribosomal footprinting of MDA-Parental and MDA-LM2

first: Hani

last: Goodarzi

department: Biochemistry and Biophysics

summary: We used Illlumina Artseq Ribo Profile to perform ribosome footprinting on the above cell lines

overall_design: We followed the manufacturer's protocol: Cells were treated with cycloheximide (0.1 mg/ml) to inhibit translation; total RNA(tt) and ribosome-protected RNA fragments (RPF) were isolated and library preped for sequencing simultaneously.

first: H

last: Nguyen

first: S

last: Tavazoie


title: Ribosomal footprinting of CN34-Parental and CN34-LM1a


title: Ligation-Free Ribosome Profiling Reveals Cell Type-Specific Regulation of Protein Synthesis in the Brain

city: New York

first: Peter

last: Sims

state: NEW YORK

address: 3960 Broadway blvd

institute: Columbia Medical Center

summary: Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep sequencing experiments. We report a new approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We used ligation-free ribosome profiling to identify new patterns of cell type-specific translation in the brain and tested its ability to identify translational targets of mTOR signaling in the brain.

overall_design: We report a strategy for ligation free cell-type specific ribosome profiling. Our strategy was utilized to explore cell-type specific expression in mouse brain.

first: Nicholas

last: Hornstein

first: Daniela

last: Torres

first: Sohani Das

last: Sharma

first: Guomei

last: Tang

last: Canoll


title: Ribosomal footprinting of MDA_Ctrl and MDA_Glu overexpression cell lines

overall_design: We followed the manufacturer's protocol: Cells were treated with cycloheximide (0.1 mg/ml) to inhibit translation; total RNA and ribosome-protected RNA fragments were isolated and library preped for sequencing simultaneously.


title: Transcriptome analysis of patient-derived xenograft models of HER2+ breast cancer brain metastases

status: Public on Jun 06 2016

first: Jean

last: Zhao

address: 450 Brookline Ave, SM936

institute: Dana Farber Cancer Institute

summary: To gain insights into tumor heterogeneity in anti-cancer drug responses of patient-derived xenograft models of HER2+ breast cancer brain metastases, we performed transcriptome gene expression profiling by Ion AmpliSeq™ Transcriptome sequencing that targets more than 20,000 human genes. Our data found that all anti-cancer drugs responders have significantly higher expression levels of AKT-mTOR-dependent signature genes as compared to the non-responders, suggesting that most HER2+ breast cancer brain metastases are depend on the AKT-mTOR pathway

overall_design: Gene expression profiles of five PDX samples were generated by Ion AmpliSeq Transcriptome sequencing, in duplcate, using Ion torrent Proton machine.

first: Jing

last: Ni

first: Shaozhen

last: Xie


title: Spatial organization shapes the turnover of a bacterial transcriptome

city: Cambridge

first: Xiaowei

last: Zhuang

address: 12 Oxford St

department: Chemistry and Chemical Biology

institute: Harvard University

summary: Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in  eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains  unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of  RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic  and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal  peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments  revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances  degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes.  Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the posttranscriptional  Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the post-transcriptional dynamics of mRNAs.

overall_design: We measure mRNA half-lives by measuring calibrated RNA abundances as a function of time after treatment with the transcription initiation inhibitor rifampicin.

first: Jeffrey

last: Moffitt


title: Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status

city: Rotterdam

first: Eskeatnaf

last: Mulugeta

address: Wytemaweg 80

department: Cell Biology

institute: Erasmus University Medical Center Rotterdam (Erasmus MC)

overall_design: 5 ChIP-seq samples including replicates and input

first: Raphaël

last: Margueron


title: Modulated expression of specific tRNA species drives cancer progression

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

overall_design: Refer to individual Series


title: RNA-Seq analysis of hnRNPL KO fetal liver cells

city: Montreal

first: Möröy

last: Tarik

state: Quebec

address: 110 Pine avenue West

country: Canada

institute: Insititut de Recherches Cliniques

summary: The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin(-)c-Kit(+) fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.

overall_design:
fetal liver cells from either hnRNPL wild-type or hnRNPL KO embryos were analysed for differential expression and alternative splicing by RNA-Seq.
RNA-Seq was carried out in biological triplicate for each sample type. Each sample is a single embryo.

first: Damien

last: Grapton

first: Tarik

last: Möröy

first: Charles

last: Vadnais


title: Ribosomal footprinting of MDA_Ctrl and MDA_Arg overexpression cell lines

first: Hani

last: Goodarzi

first: H

last: Nguyen

first: S

last: Tavazoie


title: T-bet recruits P-TEFb to super-enhancers to regulate T helper cell differentiation

first: Richard

last: Jenner


title: YAP/TAZ control peripheral myelination by regulating Schwann cell proliferation and the expression of laminin receptors

city: Buffalo

first: Fraser

middle: James

last: Sim

address: 3435 Main Street

department: Pharmacology

institute: University at Buffalo

summary: Myelination is essential for nervous system function. Schwann cells interact with neurons and with the basal lamina to sort and myelinate axons, using known receptors and signaling pathways. In contrast, the transcriptional control of axonal sorting and the role of mechano-transduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. Here, we describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice we show that Taz is required in Schwann cells for radial sorting and myelination. Yap is redundant with Taz as ablation of both Yap and Taz abolishes radial sorting. Yap/Taz regulate Schwann cell proliferation and transcription of basal lamina receptors, both necessary for proper radial sorting of axons, and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction  to myelin formation in Schwann cells.

overall_design: 3 cKO and 3 control wild-type mice

first: Yannick

last: Poitelon

first: C

last: Lopez-Anido

first: K

last: Catignas

last: Berti

first: M

last: Palmisano

last: Williamson

first: D

last: Ameroso

last: Abiko

first: Y

last: Hwang

first: A

last: Gregorieff

first: J

last: Wrana

last: Asmani

first: R

middle: J

first: Larry

last: Wrabetz

last: Svaren

first: Laura

middle: M

last: Feltri


title: RNAseq of microglia from Rab7 Mutants & Control and Wild-Type mice

city: Munich

first: Moritz

last: Rossner

address: Nussbaumstr. 7

department: Psychiatry

summary: We purified by magnet assisted cell sorting microglial cells from brains of adult Rab7 null mutant, aged mice and respective controls, isolated total RNA and performed RNAseq to determine the transciptome profiles.

overall_design: Examination of transcriptomes of Rab7 null mutants and control (2 replicates each) and aged mice and young controls (3 replicates each)

first: Nirmal

last: Kannaiyan

first: Mikael

last: Simons


title: Microarray analysis of gene expression profile of the inguinal fat in C57BL/6J mice after weight regain

city: Nanjing

first: Jianghuan

last: Zou

state: Jiangsu

address: Xuefu Road 12

department: MARC

institute: Nanjing University

summary: To search for genes and pathways that are involved in obesity memory, we harvested the inguinal fat after weight regain and performed gene expression profiling using mouse gene expression microarrays.

overall_design: The gene expression profiles were measured in mice which experienced high fat diet induced obesity, calorie restriction and weight regain (DIO group) and control mice (CTR group), each group have three replicates.

first: Xiang

last: Gao


title: MTHFD1 controls DNA methylation in Arabidopsis

city: Neuherberg

first: Martin

last: Groth

address: Ingolstädter Landstr. 1

department: Institute of Functional Epigenetics

institute: Helmholtz Munich

summary: In order to further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. We identified the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me, and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1.

overall_design: 2 BS-seq datasets for genome-wide analysis of differential DNA methylation, 18 BS-seq datasets for global DNA-methylation analysis, and 3 RNA-seq datasets for differential gene expression analysis.

first: Steven

middle: E

last: Jacobsen


title: NUPR1 maintains autolysosomal efflux by activating SNAP25 transcription in cancer cells

status: Public on Jun 05 2016

city: Tianjin

first: Zhenyi

last: Ma

address: 22 Qixiangtai Road

institute: Tianjin Medical University

summary: In the advanced stages of cancer, autophagy is thought to promote tumor progression through its ability to mitigate various cellular stresses. However, the details of how autophagy is homeostatically regulated in such tumors are unknown. Here, we report that NUPR1 (nuclear protein 1, transcriptional regulator), a transcriptional coregulator, is aberrantly expressed in a subset of cancer cells and predicts low overall survival rates for lung cancer patients. NUPR1 regulates the late stages of autolysosome processing through the induction of the SNARE protein SNAP25, which forms a complex with the lysosomal SNARE associated protein VAMP8. NUPR1 depletion deregulates autophagic flux and impairs autolysosomal clearance, inducing massive cytoplasmic vacuolization and premature senescence in vitro and tumor suppression in vivo. Collectively, our data show that NUPR1 is a potent regulator of autolysosomal dynamics and is required for the progression of some epithelial cancers.

overall_design: mRNA profiles of NUPR1-depleted A549 and control cells were generated by RNA-sequencing, using Illumina GAIIx or Illumina HiSeq 2000.

first: Yanchao

last: Mu

first: Xiaojie

last: Yan

first: Ding

last: Li

first: Dan

last: Zhao

first: Lingling

last: Wang

first: Xiaoyang

first: Jie

last: Yang

first: Hua

last: Zhang

first: Yanzhe

first: Yanan

last: Sun

first: Yiliang

last: Wei

first: Zhenfa

first: Xinzhong

last: Chang

first: Zhi

last: Yao

first: Shanshan

last: Tian

first: Kai

first: Lance

last: Terada

first: Zhe

last: Liu


title: The histone demethylase KDM6A controls the mammary luminal lineage through enzyme-independent mechanisms

city: Seoul

first: Sumin

last: Oh

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

department: Department of Biological Science

country: South Korea

institute: Sookmyung Women's University

summary: Mammary ducts and alveoli are composed of basal and luminal cells, with the fate and differentiation of secreting cells being controlled by hormones through specific transcription factors. This study establishes the essential role of the histone H3 lysine 27 trimethylation (H3K27me3) demethylase KDM6A (UTX) in a balanced basal and luminal cell compartment. Disproportionate formation of basal cells in the absence of KDM6A resulted in disorganized mammary ducts and alveoli and lactation failure. Mutant luminal progenitors lost their distinctive transcription factor expression pattern and acquired basal characteristics leading to a preferential expansion of this lineage. The structure of mammospheres obtained from mutant progenitors suggested they were derived from basal progenitors. The genomic H3K27me3 landscape was unaltered in the absence of KDM6A suggesting demethylase-independent mechanisms. In support of this, mammary tissue developed normally in mice expressing a catalytically inactive KDM6A. This study demonstrated that mammary luminal progenitor cells rely on UTX to stably maintain their identity and thereby establish a balance of basal and luminal cells required for a functional mammary gland.

overall_design: mRNA-seq in WT;MMTV-Cre (Control) and Kdm6af/f;MMTV-Cre(Kdm6a KO) at virgin. ChIP-seq for KDM6A, and NFIB in mammary tissues at virgin and KDM6A KO H3K27me3, H3K4me1, PR, and ER in mammary tissues at p13.

first: Kyung

last: Yoo

first: Chul

first: Keunsoo

last: Kang

last: Ge

first: Gertraud

middle: W

last: Robinson

first: Lothar

last: Hennighausen


title: Targeting MTHFD2 in Acute Myeloid Leukemia

first: Gabriela

last: Alexe

state: MA

address: 415 Main St.

department: Computational Biology and Bioinformatics

institute: Broad Institute

summary:
Purpose:  Identify new targets in acute myeloid leukemia (AML).
Methods:  MOLM-14 cells were transduced with lentivirus encoding shRNAs targeting MTHFD2 (shMTHFD2 hairpin TRCN0000036553, denoted M5) and control (LacZ, shControl TRCN0000072231). RNA from 6 samples, biological duplicates (LacZ1, LacZ2; M5-1, M5-2) and a technical replicate (LacZ3, M5-3) were sequenced as 50+50 bp paired-end reads using Illumina TruSeq strand specific library. The pool of six samples was sequenced on two lanes of an Illumina HiSeq, generating 101bp paired end reads.  The software package RSEM (Li et al., 2001) was run using Bowtie (version 1.0.0) to align the reads that passed quality filters  to the hg19 GENCODE version 17 (http://www.gencodegenes.org/releases/17.html) transcriptome and to quantify transcript abundance at isoform and gene level.
Results: MTHFD2 suppression induces AML differentiation. There was upregulation of well-validated myeloid differentiation genes and gene sets consistent with myeloid maturation.
Conclusion: Our study supports the therapeutic targeting of MTHFD2 in AML.

overall_design: mRNA profiles of  shMTHFD2 and shControl  MOLM-14 cells  were generated by deep sequencing, in triplicate, using Illumina TruSeq library.

first: Kimberly

last: Stegmaier


title: Differential RNA Interactions with Heterogeneous Nuclear Ribonucleoproteins During Vesicular Stomatitis Virus Infection

status: Public on Jun 04 2016

city: Winston-Salem

first: Adam

last: Davidson

state: NC

address: 1834 Wake Forest Road

department: Chemistry

institute: Wake Forest University

summary: Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in many processes in RNA metabolism. In addition to their functions in the nucleus, hnRNPs can function in the replication of RNA viruses in the cytoplasm. In vesicular stomatitis virus (VSV)-infected cells, several hnRNPs relocalize from the nucleus to the cytoplasm. This raises the question of whether these hnRNPs are relocalized together with their host nuclear RNAs or whether they associate with new RNAs in the cytoplasm. hnRNP A1, hnRNP C1/C2, and hnRNP K were immunoprecipitated from mock- or VSV-infected cells, and RNAs were analyzed by high content RNA sequencing.  Each hnRNP displayed a loss of interaction with cellular transcripts in favor of viral mRNAs. hnRNP A1 was preferentially associated with VSV phosphoprotein (P) mRNA; hnRNP C1/C2 was preferentially associated with VSV glycoprotein (G) mRNA, and hnRNP K bound viral transcripts in rank of abundance.

sample_organism:
Homo sapiens
Vesicular stomatitis virus

platform_organism:
Homo sapiens
Vesicular stomatitis virus

overall_design: Two experiments, 5 biological samples each. Mock infections and non-specific antibodies used as controls.

first: Douglas

last: Lyles

first: Rebecca

last: Alexander


title: Identification of CEBPD regulated genes in MCF7 cells

city: Frederick

first: Esta

last: Sterneck

state: MD

address: 1050 Boyles Street

institute: National Cancer Institute

summary:
Purpose: To determine which genes are regulated by CEBPD in MCF-7 cells.
Methods: MCF7 cells were transfected with siRNAs against CEBPD or control. After 48h transfection total RNAs from two independent experiments were extracted and subjected to deep sequencing following Illumina standard protocols.
Results: Samples were aligned to the reference human genome hg19 with good alignment ranging from 84 – 85% for both reads and very low mismatch rates <0.35%. RNA mapping statistics calculated using Picard software reported mapping of samples to 86 % mRNA bases, 63-65% unique reads, and <2.6% ribosomal reads. The aligned BAM files were imported into Partek Genome Studio v6.4 following the RNA-Seq workflow. Briefly, metadata about the samples was added followed by normalizing counts and doing differential expression analysis with ANOVA for the siNS vs siCEBPD contrasts. Differential gene list were created based on a p-value cutoff of <0.05 and fold-change of >1.5 or < -1.5. Analysis revealed that C/EBPδ supports the expression of 319 genes (downregulated by siCEBPD) and attenuates the expression of 238 genes (induced by siCEBPD). For validation, 31 genes were assessed by QPCR with mRNA samples independent of those used for mRNA-Seq, and also by silencing CEBPD with either one of two siRNA sequences. About 90% of the tested genes from the mRNA-Seq approach were validated as C/EBPδ-regulated in MCF-7 cells by QPCR.
Conclusions: Our study is the first characterization of the CEBPD transcriptome generated by RNA-seq in MCF7 cells with biological replicates.

overall_design: MCF7 cells were transfected with siRNA against CEBPD and control. 48h after total RNA was extracted followed by deep sequencing, in duplicate, using Illumina PE HiSeq2000.

first: Daniel

last: Mendoza


title: Identification of a novel PPARβ/δ / miR-21-3p axis in UV-induced skin inflammation [human miRNA-seq]

status: Public on Jun 03 2016

city: Lausanne

first: Mark

last: Ibberson

address: Genopode building

department: Vital-IT

institute: SIB Swiss Institute of Bioinformatics

summary: Although excessive exposure to UV is widely recognized as a major factor leading to skin perturbations and cancer, the complex mechanisms underlying inflammatory skin disorders resulting from UV exposure remain incompletely characterized. The nuclear hormone receptor PPARβ/δ is known to control cutaneous repair and UV-induced cancer development. Here, we describe a novel PPARβ/δ-dependent molecular cascade involving TGFβ-1 and miR-21-3p, which is activated in the epidermis in response to UV exposure. We establish that the passenger miRNA miR-21-3p, that we identify as a novel UV-induced miRNA in the epidermis, plays a pro-inflammatory function in keratinocytes, and that its high level of expression in human skin is associated with psoriasis and squamous cell carcinomas. Finally, we provide evidence that inhibition of miR-21-3p reduces UV-induced cutaneous inflammation in ex vivo human skin biopsies, thereby underlining the clinical relevance of miRNA-based topical therapies for cutaneous disorders.

overall_design: Epidermis and dermis, PPARB KO vs WT, 3-4 biological replicates per condition

first: Gwendoline

last: Degueurce

first: Ilenia

last: D’Errico

first: Christine

last: Pich

first: Frédéric

last: Schütz

first: Alexandra

last: Montagner

first: Marie

last: Sgandurra

first: Lionel

last: Mury

first: Paris

last: Jafari

first: Akash

last: Boda

first: Julien

last: Meunier

first: Roger

last: Rezzonico

first: Nicolò

last: Brembilla

last: Hohl

first: Antonios

last: Kolios

first: Günther

last: Hofbauer

first: Ioannis

last: Xenarios

first: Liliane

last: Michalik


title: Metabolic Engineering of Squalene Biosynthesis via Different Genomes Impacts Non-target Cellular Metabolomic and Transcriptomic Pathways

city: Philadelphia

first: Henry

last: Daniell

state: Pennsylvania

address: 240 South 40th street

department: Biochemistry

institute: University of Pennsylvania

summary: The impact of metabolic engineering on non-target pathways is a poorly explored question that requires deeper mechanistic investigation. Therefore, Nicotiana tabacum was engineered via the chloroplast (C), nuclear (N) or both (CN) genomes to express genes encoding FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) to promote squalene biosynthesis. SQS levels were ~4,300-fold higher in C and CN lines than in the N line, but all accumulated ~150-fold more squalene due to substrate or storage limitations. Slower growth and abnormal flowering phenotypes occurred regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: 65-120 unrelated metabolites changed > 32-fold. Profound effects of transgenesis on non-target gene expression included changes in the expression of 19,076 transcripts by > 2,000-fold in the CN line; 7,784 transcripts by > 1,400-fold in the N line; and 5,224 transcripts by > 2,200-fold in the C line. Cell cycle-associated transcripts were disproportionally repressed in all three lines, as shown by both manual and Gene Ontology analysis, and transporter-related transcripts were induced. Trends observed in transcriptomes were further validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signaling.

overall_design: RNA-seq in Nicotiana tabacum lines engineered via the chloroplast, nuclear or both genomes to express genes encoding farnesyl diphosphate synthase (FPS) and squalene synthase (SQS) to promote squalene synthesis

first: Elise

last: Van Buskirk

first: Ian

last: Silverman

first: Brian

last: Gregory


title: GPS2 cistrome analysis in BMDMs and RAW264.7 cells and epigenome comparison between WT and GPS2-knockout BMDMs.

city: Huddinge

first: Eckardt

last: Treuter

address: Hälsovägen 7-9

department: Department of Biosciences and Nutrition

country: Sweden

institute: Karolinska Institute

summary: GPS2 binding sites in BMDMs can be localized specially in enhancers (H3K27ac) and promoters (H3K27ac, H3K4me3). Upon GPS2 knock-out in BMDMs, de-repression of certain inflammatory genes occur, as accompanied by increased recruitment of H3K27ac and H3K4me3 marks.

overall_design: GPS2 cistrome was analyzed in WT and GPS2-KO BMDMs in basal conditions, and also in RAW264.7 macrophage cell line, using different home raised antibodies for GPS2. Also the epigenomic status of GPS2-KO vs WT BMDMs was analyzed using the histone markers H3K4me3 and H3K27ac.

first: T

last: Damdimopoulos

first: S

last: Goñi

first: Z

last: Huang

first: E


title: ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells (ChIP-seq)

city: Naples

first: Marco

last: Cammisa

state: Naples

address: Via Castellino Pietro, 111

country: Italy

institute: Institute of Genetics and Biophysics "A. Buzzati Traverso" (IGB-ABT)

summary: ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

overall_design: Examination of ZFP57, KAP1 and H3K9me3 occupancy in Zfp57-/-  mouse ESCs compared to the wild-type.

first: Vincenzo

last: Riso

first: Harpreet

last: Kukreja

first: Zahra

last: Anvar

first: Gaetano

last: Verde

first: Angela

last: Sparago

first: Basilia

last: Acurzio

first: Shraddha

last: Lad

first: Vincenza

last: Lonardo

first: Aditya

last: Sankar

first: Kristian

last: Helin

first: Robert

last: Feil

first: Annalisa

last: Fico

first: Claudia

last: Angelini

first: Giovanna

last: Grimaldi

first: Andrea

last: Riccio


title: ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells  (RNA-seq)

overall_design: Examination of mRNA levels in Zfp57-/- mouse ESCs compared to the wild-type.


title: Towards therapeutic application of pronuclear transfer to prevent transmission of mitochondrial DNA disease

city: London

first: Kathy

last: Niakan

address: Mill Hill

institute: The Francis Crick Institute

summary: Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Assisted reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable women who carry mtDNA mutations to have a genetically related child with a greatly reduced risk of disease. Here we report for the first time that pronuclear transplantation (PNT) between normally fertilised human zygotes provides an effective approach to preventing transmission of mtDNA disease.  We found that the procedures previously used to perform PNT between abnormally fertilized human zygotes are highly inefficient when applied to those that undergo normal fertilization.  We have therefore developed an alternative approach based on transplanting PN shortly after completion of the second meiotic division rather than shortly before onset of the first mitosis.  This approach promotes highly efficient development to the blastocyst stage without affecting nuclear genome integrity. Furthermore, the expression profile of genes encoded by the nuclear and mitochondrial genomes was indistinguishable from unmanipulated control embryos. Importantly, levels of mtDNA transferred with the nuclear genome are below the threshold for mtDNA disease. Together these data indicate that transplantation of pronuclei early in the first cell cycle holds promise as a safe and effective approach to preventing transmission of mtDNA disease.

overall_design:
Single-Cell RNA-seq analysis of embryos generated by pronuclear transfer and unmanipulated control embryos

The relationship between single cell samples and the embryo from which they were derived is indicated in the sample 'characteristics: sample type' field.

first: Louise

last: Hyslop

first: Paul

last: Blakeley

first: Lyndsey

last: Craven

first: Norah

last: Fogarty

first: Jessica

last: Richardson

first: Elpida

last: Fragouli

first: Laura

last: Irving

first: Meenakshi

last: Choudhary

first: Mahdi

last: Lamb

first: Samer

last: Alfarawati

first: Dimitrios

last: Kalleas

first: Dagan

last: Wells

first: Alison

middle: P

last: Murdoch

first: Douglass

last: Turnbull

first: Mary

last: Herbert


title: Identification of a novel PPARβ/δ / miR-21-3p axis in UV-induced skin inflammation

first: Mark

last: Ibberson

overall_design: Refer to individual Series


title: ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells (BS-seq and RRBS)

first: Marco

last: Cammisa

overall_design: Examination of methylation levels in Zfp57-/- mouse ESCs compared to the wild-type.

first: Vincenzo

last: Riso

first: Harpreet

last: Kukreja

first: Zahra

last: Anvar

first: Gaetano

last: Verde

first: Angela

last: Sparago

first: Basilia

last: Acurzio

first: Shraddha

last: Lad

first: Vincenza

last: Lonardo

first: Aditya

last: Sankar

first: Kristian

last: Helin

first: Robert

last: Feil

first: Annalisa

last: Fico

first: Claudia

last: Angelini

first: Giovanna

last: Grimaldi

first: Andrea

last: Riccio


title: ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells


title: RNA-sequencing time course of Human Intestinal Epithelial Cells (HIECs) following knockdown of miR-30bcd using complementary locked nucleic acids

city: Chapel Hill

first: Bailey

last: Peck

address: 120 Mason Farm Rd, 5100H Genetic Medicine Bldg

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: To gain mechanistic insight into our finding that knockdown of miR-30 in HIECs and Caco-2 cells resulted in increased SOX9 mRNA, but decreased SOX9 protein expression, we performed RNA-sequencing on LNA30bcd-treated HIECs. We found 2440 significantly increased genes and 2651 significantly decreased genes across three time points (24 hours, 48 hours, and 72 hours post-transfection) relative to mock transfected cells. The up-regulated genes are highly enriched for both predicted miR-30 targets as well as genes in the ubiquitin-proteasome pathway. Inhibition of miR-30 also led to significantly reduced IEC proliferation and an increase in markers of enterocyte differentiation.

overall_design: We evaluate transcriptional changes across time following knockdown of miR-30 family members in HIECs. We include 3 replicates each of Mock and LNA30bcd treated cells at 3 time points, 24H, 48H, and 72H post-transfection.

first: Praveen

last: Sethupathy

first: Pauline

last: Lund


title: Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development

city: Brookline

first: Yuting

last: Liu

address: 82 Walnut St. Unit 3

department: Department of Genetics

institute: Harvard Medical School

summary: Upon fertilization, parental chromatin undergoes extensive reprogramming to generate the highly dynamic chromatin of the inner cell mass (ICM) cells from which an organism is derived. How the chromatin regulatory landscape is established during this process has remained a mystery due to the technical difficulties in chromatin analysis using limited cell numbers. Using a low-input DNase I sequencing (liDNase-seq) method, we generated DNase I-hypersensitive site (DHS) maps of mouse preimplantation embryos. We found that the DHSs are progressively established during development, with Oct4 contributing to a drastic gain of DHSs at the 8-cell stage. In addition, single nucleotide polymorphism analysis revealed that imprinted gene promoters harbor allele-specific DHSs before the onset of allelic gene expression. Furthermore, Nfya is required for 2-cell promoter DHS formation and zygotic genome activation. Thus, our study not only reveals chromatin regulatory landscapes, but also identifies key transcription factors important for DHS establishment in mammalian embryos.

overall_design: In this study, we modified scDNase-seq method to achieve de novo genome-wide DHS mapping using as few as 30 cells. Using this low-input DNase-seq (liDNase-seq) method, we have generated DHS maps of mouse preimplantation embryos from 1-cell to the morula stage, revealing the dynamics of chromatin accessibility during the natural developmental process. We constructed a total of 32 liDNase-seq datasets, including: two 1-cell embryo libraries, two 2-cell embryo libraries, two 4-cell embryo libraries, two 8-cell embryo libraries, two morula embryo libraries, two mouse ESC libraries, two 14 day growing oocyte library, two sperm library, two maternal PN3 pronuclei library, two paternal PN3 pronuclei library, two maternal PN5 pronuclei library, two paternal PN5 pronuclei library, two siRNA control 2-cell embryo libraries, two Nfya KD (siNfya#1 and siNfya#2) 2-cell embryo libraries, two siRNA control 8-cell embryo libraries and two Oct4 KD 8-cell (siOct4#1 and siOct4#2) embryo libraries. Five RNA-seq datasets were generated including two siRNA control 2-cell embryo libraries, two Nfya KD (siNfya#1 and siNfya#2) 2-cell embryo libraries and one 1-cell embryo library.

first: Falong

last: Lu

first: Yyuting

first: Azusa

last: Inoue

first: Tsukasa

last: Suzuki

first: Keji

last: Zhao

first: Yi

last: Zhang


title: Impairment of DNA methylation maintenance is the main cause of global demethylation in naïve ES cells

city: Nijmegen

first: Hendrik

middle: G

last: Stunnenberg

address: Geert Grooteplein 28

department: Molecular Biology

institute: Radboud University

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


title: CTCF binding profiles in MEF cells

first: Tae-Young

last: Roh

state: --- Select One ---

address: 52 Ewhayeodae-gil, Seodaemun-gu

department: Life Sciences

institute: Ewha Womans University

summary: We sequenced CTCF binding sites in Akt1+/+ or Akt1-/- mouse embryonic fibroblast (MEF) cell line.

overall_design: The information of CTCF binding sites in wildtype (WT) and Akt1-/- MEF cells was generated by deep sequencing, using Illumina GAIIx.

first: Chang-Pyo

last: Hong

first: Seong-Hye

last: Seo

first: Hyehyun

last: Min

first: Kyoung-Ah

last: Kong

first: Ji-Yeon

last: Lee

first: Sun

last: Bae

first: Myoung

middle: H

last: Kim