title: High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism

city: Montreal

first: Mahmoud

last: Aarabi

state: Quebec

address: 1001 Boulevard Décarie

department: Human Genetics

country: Canada

institute: McGill University

summary: Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodelled during spermatogenesis. While high dose folic acid supplementation (up to ten times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of six months of high dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1.  Our data reveal alterations of the human sperm epigenome associated with high dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR.

overall_design: Reduced representation bisulfite sequencing of 28 human sperm samples before and after 6 month of high dose folic acid supplementation.

first: Maria

middle: C

last: San Gabriel

first: Donovan

last: Chan

first: Nathalie

middle: A

last: Behan

first: Maxime

last: Caron

first: Tomi

last: Pastinen

first: Guillaume

last: Bourque

first: Amanda

middle: J

last: Macfarlane

first: Armand

last: Zini

first: Jacquetta

last: Trasler


title: RNA-seq analysis of acute Rb1 deletion in adult mouse tissues

status: Public on Sep 11 2015

city: Charlestown

first: Robert

last: Morris

state: Massachusetts

address: 149 13th Street

country: USA

institute: Mass. General Hospital Cancer Center

type: Expression profiling by high throughput sequencing

summary: Abstract:The pRb tumor suppressor protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb-loss are largely unexplored. We have acutely ablated Rb in adult mice and conducted quantitative analysis of RNA and proteomic changes in colon and lung, where Rb-loss was sufficient or insufficient to induce ectopic proliferation respectively. As expected, RbKO caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but unexpectedly their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb-loss are coupled with proliferation, but uncoupled from transcription. The proteomic changes in common between RbKO tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and OXPHOS function. RBKO cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb-loss altered mitochondrial pyruvate oxidation from 13C-Glucose through the TCA cycle in both cells and mouse tissues. Consequently, RBKO cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

overall_design:
Design: Two tissues (lung and colon) from adult control (Rb1 wild type) or variable (Rb1 acute knockout). We examined the consequences of pRb loss in an in vivo model.  Mice were bred to introduce a tamoxifen inducible form of Cre recombinase (ROSA26Sortm1(cre/Esr1)Tyj ) into the genetic backround of either Rb wild type (Rb+/+) or Rb floxed (Rbf/f) alleles. These animals were then injected with tamoxifen, ablating Rb in Rbf/f mice and generating RbKO tissues. Lung and colon tissues were harvested 96 hrs after the tamoxifen injections and analyzed. Each cohort used 3 biological replicates.
Methods: RNA extraction: Ninety six hours following the last injection of tamoxifen mice were processed were sacrificed and dissected tissues flash frozen in liquid nitrogen for RNA extraction. RNA was extracted using the TRIzol-choloroform method and then DNase treated. RNA quality was validated using a bioanalyzer. Library construction and RNA-squencing was performed at the MGH NextGeneration Sequencing facility. Three mice were used per genotype, taking matched tissues from each mouse.
Methods: Alignment: Sequenced libraries were mapped to the mouse genome (mm10) using TOPHAT (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016). A RefSeq GTF file was used to annotate the mapped transcripts. FPKM matrixes and differentially regulated gene lists were generated using the Cufflinks-Cuffmerge-Cuffdiff pipeline (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016).

first: Brandon

middle: N

last: Nicolay

first: Nicholas

last: Dyson

first: Filippos

last: Kottakis


title: Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

status: Public on Sep 10 2015

city: Ipswich

first: Guoqiang

last: Zhang

address: 240 County Road

institute: New England Biolabs Inc

type: Methylation profiling by high throughput sequencing

summary: Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity and was rescued by transfection of DNMT1. Decrease in DNMT1 specific activity led to hypomethylation of the genome. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, 14-3-3 overexpression also resulted in enhanced cell invasion. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNMT1 level, DNA methylation and altered gene expression that contributes to cell invasion.

overall_design: Examine of the DNA methylation and RNA expression profiles of stable NIH3T3 clones overexressing the 14-3-3ε homologue

first: Zhang

last: Guoqiang

first: Pradhan

last: Sriharsa


title: Genome-wide DNA methylation analyses by the MeDIP assay

city: Baltimore

first: Minoru

last: Ko

state: MD

address: 251 Bayview Blvd, Suite 100, 10C

department: National Institute on Aging

institute: NIH

summary: We analyzed the genome-wide DNA methylation in Zscan4 overexpressing ES cells. Zscan4 overexpression induced slight DNA demethylation in telomere and major satellite regions. Subsequent genome-wide analysis of non-repeated regions revealed the significant reduction of DNA methylation at Zscan4-dependent hyperacetylation sites. This result indicates that Zscan4 is not only a marker of the Zscan4+ state of ES cells, but also indispensable for the dramatic epigenetic modifications occurring in the Zscan4+ state.

overall_design: DNA methylation in Zscan4 overexresssing ES cells

first: Li

last: Xin

first: Alexei

last: Sharov

first: Yong

last: Qian


title: ChIP-seq assays for H3K9me2

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We analyzed the genome-wide chromatin states in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-) by using FACS-sorted MC1-ZE7 ES cells. H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells. These results suggested that the heterochromatin is activated in Zscan4 positive state.

overall_design: H3K9me2 in Zscan4 positive and negative cells

first: Tomohiko

last: Akiyama

first: Mayumi

last: Oda

last: Sharav

first: Yulan

last: Piao


title: Rif1 binds to G-quadruplexes and suppresses replication over a long distances

city: Tokyo

first: Yutaka

last: Kanoh

address: 2-1-6 Kamikitazawa, Setagaya-ku

department: Genome Medicine

country: Japan

institute: Tokyo Metropolitan Institute of Medical Science

summary: Rif1 regulates replication timing and repair of double-stranded DNA breaks. Using Chromatin-immunoprecipitation-Sequencing method, we have identified 35 high-affinity Rif1 binding sites in fission yeast chromosomes. Binding sites, preferentially located to the vicinity of dormant origins, tended to contain at least two copies of a conserved motif, CNWWGTGGGGG, and base substitution within these motifs resulted in complete loss of Rif1 binding and activation of late-firing or dormant origins located as far as 50 kb away. We show that Rif1 binding sites adopt G-quadruplex-like structures in vitro in a manner dependent on the conserved sequence as well as on other G-tracts, and that the purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds to G-quadruplex-like structures at selected intergenic regions to generate local chromatin structures that may exert a long-range suppressive effects on origin firing.

overall_design: ChIP-Seq profiles of Rif1 and DNA replicaiton (BrdU-incorporation) vs Input in wildt type, rap1∆, taz1∆, Rif1BS mutants and rif1∆

first: Seiji

last: Matsumoto

first: Rino

last: Fukatsu

first: Naoko

last: Kakusho

first: Nobuaki

last: Kono

first: Claire

last: Renard-Guillet

first: Keisuke

last: Iida

first: Kazuo

last: Nagasawa

first: Katsuhiko

last: Shirahige

first: Hisao

last: Masai


title: ChIP-seq assays for H3K27ac

first: Minoru

last: Ko

summary: By using FACS-sorted MC1-ZE7 ES cells, we analyzed the genome-wide distribution of H3K27ac and DNA methylation in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-). H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells, but not Em- cells. These results suggested that the Zscan4 state takes "open" chromatin conformation in ES cells.

overall_design: H3K27ac in Zscan4 positive and negative cells


title: High-throughput sequencing of human macrophages infected with Mycobacterium abscessus Smooth and Rough variants

status: Public on Sep 09 2015

city: Dublin

first: Brendan

middle: J.

last: Loftus

address: Conway Institute, University College Dublin

department: School of Medicine & Medical Science

country: Ireland

institute: University College Dublin

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Genome-wide transcriptome analyses by the RNA-seq method

summary: We performed the whole transcriptome analysis in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-) by using FACS-sorted MC1-ZE7 ES cells.

overall_design: Whole RNA-seq in Zscan4 positive and negative cells

first: Alexei

last: Sharov

first: Dawood

last: Dudekula

last: Qian


title: The methylome of Alu repeats in primates

city: Badalona

first: Miguel

middle: A.

last: Peinado

address: Can Ruti Campus

country: Spain

institute: IGTP

summary: Next Generation Sequencing of Unmethylated Alu (NSUMA) interrogation of more than 130,000 individual Alus for differential methylation with concomitant analysis of copy number variations applied to the study of hypomethylation in primates.

sample_organism:
Gorilla gorilla
Pan troglodytes
Pongo pygmaeus
Homo sapiens

overall_design: 3 replicates of Gorilla gorilla, Pan troglodytes, Pongo pygmaeus and Homo sapiens were studied.

first: Berta

last: Martín

first: Mireia

last: Jordà

first: Sergi

last: Lois

first: Izaskun

last: Mallona


title: Zscan4 mediates transient remodeling and transcriptional burst of heterochromatin in mouse embryonic stem cells

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

Repetitive sequences such as telomeres, centromeres, and retrotransposons are packed in permanently-condensed and transcriptionally-silenced heterochromatin, whose maintenance is critical for the genome stability. We have recently found that mouse ES cells occasionally go through a unique cell state marked by the transient expression of Zscan4, which plays a key role in long-term genome stability. Here we report our unexpected and counterintuitive finding that the Zscan4+ state is accompanied by a Zscan4-mediated rapid activation and repression of heterochromatin: the burst of transcription from heterochromatin coincided with its transient histone hyperacetylation and DNA demethylation, the clustering of pericentromeric heterochromatin in the perinucleolar region, and the accumulation of chromatin remodeling complexes for both activation (HATs, SWI/SNF) and repression (HDACs, LSD1/KDM1A, NuRD) on heterochromatin. Overall, these findings suggest that ES cells maintain their extraordinary genome stability and immortality by transiently taking extreme measures – a usually forbidden transition to the activated state of heterochromatin.


title: Poly(A)-specific ribonuclease (PARN) mediates 3' end maturation of the telomerase RNA component

status: Public on Sep 08 2015

first: Patrick

last: Cahan

address: 733 North Broadway, MRB 653

department: ICE and Biomedical Engineering

institute: Johns Hopkins University School of Medicine

summary: Mutations in the poly(A) ribonuclease (PARN) gene cause telomere diseases including familial idiopathic pulmonary fibrosis (IPF) and dyskeratosis congenita (DC)1,2, but how PARN deficiency impacts telomere maintenance is unclear. Here, using somatic cells and induced pluripotent stem (iPS) cells from DC patients with PARN mutations, we show that PARN is required for the 3′ end maturation of the telomerase RNA component (TERC). Patient cells as well as immortalized cells in which PARN is disrupted show decreased levels of TERC. Deep sequencing of TERC RNA 3′ termini reveals that PARN is required for removal of posttranscriptionally acquired oligo(A) tails that target nuclear RNAs for degradation. Diminished TERC levels and the increased oligo(A) forms of TERC are normalized by restoring PARN, which is limiting for TERC maturation in cells. Our results reveal a novel role for PARN in the biogenesis of TERC, and provide a mechanism linking PARN mutations to telomere diseases.

overall_design: mRNA sequencing of fibroblasts, induced pluripotent stem cells, and 293 cell line.

first: Diane

middle: H

last: Moon

first: Suneet

last: Agarwal


title: The methylome of Alu repeats in colon cancer (NSUMA)

summary: Next Generation Sequencing of Unmethylated Alu (NSUMA) interrogation of more than 130,000 individual Alus for differential methylation with concomitant analysis of copy number variations applied to the study of hypomethylation in colorectal cancer.

overall_design: normal colon tissues, matched primary tumors, blood and colon derived cell lines

first: Anna

last: Díez-Villanueva


title: The methylome of Alu repeats in colon cancer

type: Genome variation profiling by genome tiling array


title: Association of Taf14 with acetylated histone H3 directs the DNA damage response and gene transcription

city: Chapel Hill

first: Erin

middle: K

last: Shanle

state: NC

address: 3010 Genetic Medicine, 120 Mason Farm Road

department: Biochemistry and Biophysics

institute: University of North Carolina at Chapel Hill

summary: We sequenced mRNA from triplicate log-phase cultures of BY4741 (WT) transformed with pRS313-HA3-SSN6 and taf14D transformed with pRS313-HA3-SSN6 (empty vector), full-length pRS313-TAF14-HA3-SSN6, or pRS313-taf14W81A-HA3-SSN6 cultured in synthetic complete media lacking histidine.

overall_design: Examination of changes in gene expression when the YEATS domain of Taf14 is mutated so it cannot bind acetyl-H3.

first: E

first: B

middle: D

last: Strahl


title: Mutual Regulation between Satb2 and Fezf2 Promotes Subcerebral Projection Neuron Identity in the Developing Cerebral Cortex

city: Santa Cruz

first: Bin

last: Chen

state: CA

address: 1156 High St.

department: MCD Biology

institute: University of California Santa Cruz


title: Functional Evidence Implicating Chromosome 7q22 Haploinsufficiency in Myelodysplastic Syndrome Pathogenesis

city: Memphis

first: Jeffery

last: Klco

state: TN

address: 262 Danny Thomas Place

department: Pathology

institute: St Jude Children's Research Hospital

summary:
Purpose: 5A3+/del mice, which have a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22, exhibit hematopoietic stem cell (HSC) abnormalities that may contribute to myelodysplastic syndrome (MDS) pathogenesis. These defects are cell autonomous. The goal of this study is to compare transcriptome profiling (RNA-seq) data obtained from HSC and multipotent progenitors (MPP) isolated from 5A3+/del mice and their wildtype littermates, in order to identify differentially expressed genes and pathways that may contribute to the phenotype.
Methods: Total RNA was isolated from CD150hi-HSC, CD150lo-HSC and CD150neg-MPP from wildtype and 5A3+/del mice. 10ng of total RNA was converted into double-stranded cDNA using the Ovation RNA Amplification System V2 (NuGen, CA), and the amplified cDNA products were then used to generate RNA-seq libraries using the TruSeq RNA Sample Preparation Kit v2 reagents (Illumina, CA) with 10 PCR amplification cycles. Library quality and quantity were assessed by the Agilent DNA1000 Chip (Agilent, CA) and qPCR (Kappa Biosystems Inc, MA). 10 pM of each library was sequenced using Illumina SBS chemistry at 2 x 100 bp reads on the HiSeq2000 (Illumina®, CA). The RNA-Seq paired-end reads were mapped to the mouse mm9 genome using an in-house mapping and quality assessment pipeline. The expression of each gene was estimated by the mean coverage of the highest-covered coding exon. Genes with low expression level (<10) across all samples were filtered out, followed by quantile normalization. Differential expression analysis was performed using limma with estimation of false-discovery rate. Gene Set Enrichment Analysis was used to assess pathway enrichment.
Results:Transcriptome (RNA-Seq) and Taqman quantitative real-time PCR analyses revealed a ~50% reduction in the expression of multiple genes and of the long intergenic non-coding RNA 503142E22Rik within the 5A3 interval in mutant HSC and MPP. Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data from 5A3+/del HSCs further demonstrated reduced expression of multiple gene sets related to oxidative phosphorylation (OXPHOS) that are similarly down-regulated in the early stages of human therapy-induced MDS and AML.
Conclusions: Our study revealed that genes involved in OXPHOS were down-regulated in 5A3+/del HSC, and this finding provided novel insights into the impact of chromosome 7 deletions on the pathogenesis of MDS.

overall_design: Transcriptome profiling (RNA-seq) data of HSC and MPP isolated from 5A3+/del mice and their wildtype littermates were generated by sequencing, in quintuplicate, using Illumina SBS chemistry at 2 x 100 bp reads on the HiSeq2000 (Illumina®, CA)

first: Jasmine

last: Wong

first: Kelley

middle: M

last: Weinfurtner

first: Joy

last: Nakitandwe

first: Jing

last: Ma

first: James

middle: R

last: Downing

first: Kevin

last: Shannon


title: High-throughput RNA-sequencing of human macrophages infected with Mycobacterium abscessus Smooth and Rough variants

first: Brendan

last: Loftus

summary: Mycobacterium abscessus is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. We sampled the small RNA (sRNA) transcriptome of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 hours post-infection (hpi) using RNA-seq. MAB-S elicited a more robust transcriptional response at the miRNA level, reflecting higher cytokine levels in culture supernatants. However, and a direct comparison identified no differentially expressed miRNAs between MAB-R- and MAB-S-infected cells. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes.

overall_design: THP-1-derived macrophages were infected in parallel with the MAB-R and MAB-S morphotypes. Poly-A selected RNAs were purified and sequenced at 1, 4 and 24 hours post-infection, and compared with uninfected controls.

first: Adam

last: Dinan


title: High-throughput small RNA-sequencing of human macrophages infected with Mycobacterium abscessus Smooth and Rough variants

overall_design: THP-1-derived macrophages were infected in parallel with the MAB-R and MAB-S morphotypes. Size-selected small RNAs were purified and sequenced at 1, 4 and 24 hours post-infection, and compared with uninfected controls.


title: An essential role for the Gai2 protein in Smoothened-stimulated mammary epithelial cell proliferation

city: Houston

first: Chad

last: Shaw

state: TX

address: 1 Baylor Plaza

department: Molecular and Human Genetics

institute: BCM

summary: Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance.  Smoothened (SMO), the primary effector of Hh signaling, is expressed ectopically in human breast cancer, as well as in other cancers.  Constitutive activation of SMO in mouse mammary glands leads to paracrine stimulation of proliferation, as well as hyperplasia.  In canonical signaling, SMO functions via GLI transcription factor activation.  However, recent data from Drosophila and mammalian cell lines indicate that SMO can function non-canonically as a G-protein coupled receptor (GPCR) by coupling to heterotrimeric G proteins, particularly those in the pertussis toxin (PTX)-sensitive G-alpha-i (Gai) class.  Whether SMO functions as a GPCR in mammalian tissues in vivo is not known.  Using genetically modified mouse models, we demonstrate here that SMO-induced stimulation of proliferation is PTX sensitive, and requires Gai2, but not Gai1 or Gai3.  Our findings provide evidence for a non-canonical GPCR function of activated SMO in vivo, a finding that may have clinical significance given that most SMO-targeted agents were selected based largely on their ability to block canonical GLI-mediated transcription.

overall_design: Primary mammary epithelial cell RNA was deep-sequenced from mT-mG/SmoM2;MMTV-Cre (EGFP), mT-mG/SmoM2;MMTV-Cre (tdTomato), and mT-mG/SmoM2;+ cells to examine the effects of SmoM2 overexpression in the mammary gland.

first: Michael

middle: T

last: Lewis


title: Effect in HCT116 cells of 3hr cortistatin A treatment on gene expression.

status: Public on Sep 25 2015

city: Cambridge

first: Matthew

middle: D.

last: Shair

state: MA

address: 12 Oxford Street

department: Chemistry and Chemical Biology

institute: Harvard University

summary:
We characterized the marine natural product cortistatin A (CA) as an inhibitor of CDK8 to determine whether pharmacologic inhibition of CDK8 regulates super-enhancer function and inhibits AML proliferation.
In this series, we examine the transcriptional effect on insensitive HCT116 cells of 3hrs exposure to CA.

overall_design: HCT116 cells were treated in triplicate with either DMSO or CA for 3hrs after which RNA was harvested and prepared for RNA sequencing to assess transcriptional changes.

first: Henry

middle: E

last: Pelish

first: Brian

middle: B

last: Liau

first: Aaron

last: Odell

first: Robin

last: Dowell

first: Zachary

last: Poss

first: Dylan

last: Taatjes

first: Madeleine

last: Lemieux


title: Ago-APP: a GW peptide-based Argonaute affinity capture strategy

city: Regensburg

first: Judith

last: Hauptmann

address: Universitaetsstrasse 31

country: Germany

institute: University of Regensburg

summary: During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs or target mRNAs. We refer to our method as Ago protein Affinity Purification by Peptides (Ago-APP).

overall_design: Comparison of small RNA lengths in total RNA and APP-enriched RNA samples

first: Norbert

last: Eichner

first: Marc

last: Urban

first: Stefanie

last: Sprunck

first: Gunter

last: Meister


title: Comparison of endogenous tRNA expression levels from cells expressing and sgRNA from either a U6 or tRNA promoter

city: Durham

middle: Lee

last: Mefferd

address: 408 Andrews Road

department: Molecular Genetics and Microbiology

institute: Duke University

summary: The goal of this study was to compare the endogenous tRNA expression levels from cells transduced with a lentiviral vector encopding Cas9 and an sgRNA from a U6 or a Glutamine tRNA promoter. Using high-throughput sequencing methods (Illumina Hi-Seq 2000) we show that endogenous tRNA expression levels are not perturbed by expression of an sgRNA from a human tRNA.

overall_design: tRNA expression profiles were generated from 2 different transduced polyconal cell lines.

first: Bryan

last: Cullen


title: ChIP-seq for Satb2 in E15 cortex

first: Bin

last: Chen

summary: The goal of the study was to identify the binding site of SATB2 in wild-ype cortex by performing ChIP-seq using SATB2 antibody. E15 cortical tissues were dissected, lysed and fixed. Chromatin was prepared by sonication. Sequences bound by SATB2 protein was precipitated using a SATB2 antibody. Sequencing was performed on Illumina Genome Analyzer II. SATB2 binding peaks were called using MACS.

overall_design: ChIP for Satb2, followed by sequencing on Illumina Genome Analyzer II platform

first: William

middle: L

last: McKenna

first: Christian

middle: F

last: Ortiz-Londono

first: Thomas

last: Mathew

first: Sol

last: Katzman


title: The alteration of 5-hydroxymethylcytosine modification contributes to ischemic brain injury [5hmC-seq]

city: Atlanta

first: Peng

last: Jin

state: Georgia

address: 615 Michael St., Rm 325

institute: Emory University

summary: Epigenetic modifications, such as cytosine methylation and histone modification, have been shown involved in the pathology of ischemic brain injury. Recent works have implicated 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) through the oxidation by Ten-Eleven Translocation (TET) enzymes, in DNA methylation-related plasticity. In this study we show that 5hmC abundance could be induced to increase by ischemia injury.  Genome-wide profiling of 5hmC identified differentially hydroxymethylated regions (DhMRs) associated with ischemic injury and DhMRs were found enriched among the genes involved in cell junction, neuronal morphogenesis and neurodevelopment. These data together suggest that 5hmC modification could serve as a potential therapeutic target for the treatment of ischemic stroke.

overall_design: To determine the genome-wide 5hmC distribution in both ischemic injury (I/R) and control mice (C57BL/6), we employed a previously established chemical labeling and affinity purification method, coupled with high-throughput sequencing (Song et al, Nature Biotechnology, 2011). The ischemic or matched control brain tissues from three pairs of ischemic mice and control mice were used for the analyses.

first: Xingshun

last: Xu


title: RNA-seq of postnatal day 0 (P0) wild-type and Satb2-/- cortices

summary: The goal of the study was to compare gene expression of P0 wild-type and P0 Satb2-/- cortices. Total RNAs were isolated from P0 cortices dissected from wild-type and Satb2-/- mice (n=3 for each genotype), following Qiagen RNAeasy kit instruction.Sequence libraries were made following Illumina RNA TruSeq library preparation guide.The libaries were pair-end sequenced (50nt per end). Differentially expressed genes were identified by DESEQ.

overall_design: Total RNAs were isolated from P0 cortices (3 control and 3 mutants), and sequenced on Illumina Genome Analyzer


title: Digital Restriction Enzyme Analysis of Methylation (DREAM) of SW48 colon cancer cell line genomic DNA.

city: Camden

first: Jaroslav

last: Jelinek

state: NJ

address: 403 Haddon Avenue

department: Research Labs

institute: Coriell Institute for Medical Research

summary: We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.

overall_design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SW48 colon cancer cell line genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.

first: Jiali

last: Si

first: Rong

last: He

first: Yue

last: Lu

first: Shoudan

last: Liang

first: Jozef

last: Madzo

first: Matteo

last: Cesaroni

first: Louis

last: Ramagli

first: Jean-Pierre

last: Issa


title: Genome-wide DNA methylation analyses by the HELP assay

first: Minoru

last: Ko

summary: The HELP tagging assay was performed on purified genomic DNAs. The protocol was modified for NEBNext Multiplex Oligos for Illumina (NEB) from the original protocol4 (http://wasp.einstein.yu.edu/index.php/Protocol:HELP_tagging). Briefly, genomic DNA was digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. AS and AE adapters were prepared by annealing two oligo DNAs separately. The first Illumina AE adapter was ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking genomic DNA sequence. An A-overhang was created, allowing the ligation of the second Illumina AS adapter. This created both AE-insert-AS products and AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we selectively enriched for the AE-insert-AS product. PCR amplification by NEBNext Multiplex Oligos was performed to generate a single-sized amplicons for Illumina sequencing.

overall_design: DNA methylation in Zscan4 positive and negative cells

first: Mayumi

last: Oda

first: Miki

last: Sakota

first: Yulan

last: Pian

first: Alexei

last: Sharov

first: Yong

last: Qian


title: Knockout mice reveal an essential role for Epithelial splicing regulatory proteins (Esrps) in mammalian development and epithelial splicing in vivo

city: Los Angeles

first: Yi

last: Xing

address: 650 Charles E. Young Dr. South

department: Department of Microbiology, Immunology, & Molecular Genetics

institute: UCLA

summary: Tissue- and cell-type specific regulators of alternative splicing (AS) are an essential layer of posttranscriptional gene regulation necessary for normal cellular function, patterning, and development. Here we report the Epithelial splicing regulatory proteins (Esrps) are required for patterning of multiple organs, with loss of both paralogs, Esrp1 and Esrp2, resulting in increasingly severe phenotypes. Global profiling of the Esrp splicing regulatory network from total epidermis revealed varied splicing sensitivity of Esrp targets upon loss of Esrp1 or double knockout. This may explain the progressive phenotypes seen in Esrp knockout mice, and these mice provide a unique genetic tool to evaluate functional consequences of epithelial splicing events in vivo.

overall_design: RNA from purified total epidermis (basal keratinocyte layer to cornified layer) of E18.5 mouse embryos were harvested by Trizol extraction. (Esrp1+/+, Esrp2-/- (n=2), Esrp1-/-, Esrp2+/+ (n=3), Esrp1-/-, Esrp2+/- (n=2), and Esrp1-/-, Esrp2-/- (n=2). 1 ug of total RNA was used for  for RNA-seq library preparation using the TruSeq™ Stranded mRNA LT Sample Prep Kit (Illumina). 100x2 bp paired-end RNA-seq reads were generated on a HiSeq 2000 sequencer.

first: Russ

last: Carstens


title: Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

status: Public on Sep 07 2015

city: Worcester

first: Isana

last: Veksler-Lublinsky

address: 373 Plantation Street

department: Molecular Medicine

institute: University of Massachusetts Medical School

summary: MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA population associated with ALG-1(anti) complexes in vivo. alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation.

overall_design: Deep-sequencing was performed on cDNA libraries made from total RNA and RNA immunoprecipitated with ALG-1 from mixed-staged populations of three strains: three biological replicates from wild-type animals and two biological replicates each from alg-1(ma192) and alg-1(ma202) mutant animals. In addition, deep-sequencing was performed on cDNA libraries made from L2-staged total RNA in two biological replicates from wildtype and alg-1(ma202) animals and one biological replicate of alg-1(ma192).

first: Anna

middle: Y

last: Zinovyeva

first: Victor

last: Ambros


title: Transcriptional plasticity promotes primary and acquired resistance to BET bromodomain inhibition

city: Stuttgart

first: Philipp

last: Rathert

state: Germany

address: Allmandring 31

department: Biochemistry

institute: University Stuttgart

summary: Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukemia (AML), BET inhibitors are being explored as promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced hematologic malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukemia, we performed a chromatin-focused shRNAmir screen in a sensitive MLL/AF9; NrasG12D‑driven AML model, and investigated dynamic transcriptional profiles in sensitive and resistant murine and human leukemias. Our screen reveals that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodeling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukemias regardless of their sensitivity, resistant leukemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signaling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic ChIP- and STARR-seq enhancer profiles reveal that BET-resistant states are characterized by remodeled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signaling as a driver and candidate biomarker of primary and acquired BET resistance in leukemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-Seq of DMSO- or JQ1-treated cancer cell lines; ChIP-seq for H3K36me3 and H3K27me3 in a leukemia cell line treated either with DMSO or JQ1, ChIP-seq for H3K27ac in resistant and sensitive mouse and human leukemia. Functional enhancer mapping (STARR-seq) in K-562 treated either with DMSO or JQ1.

first: Tobias

last: Neumann

first: Matthias

last: Muhar

first: Johannes

last: Zuber

first: Felix

last: Muerdter

first: Alexander

last: Stark

first: Jae-Seok

last: Roe

first: Christopher

last: Vakoc


title: Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale

city: Chicago

first: Dali

last: Han

state: IL

address: 5801 South Ellis Avenue

department: Department of Chemisty

institute: University of Chicago

summary: Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for the whole-genome analysis of 5fC, based on a selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

overall_design:
Utilization of cyclization-enabled C-to-T transition of 5fC (hence “fC-CET”) to obtain genome-wide map of 5fC at single-base resolution
WT and TdgKO mES cell lines.  Two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI), two pull-down output samples.

first: Bo

last: Xia

first: Xingyu

last: Lu

first: Chuan

last: He

first: Chengqi

last: Yi


title: AMPK epidermal inhibition promotes HuR cytoplasmic localization eliciting post-transcriptional inflammatory response in psoriasis

city: Montpellier

first: Geneviève

last: Garcin

address: UM2. Place Eugene Bataillon. Bat 24.

country: France

institute: CNRS UMR5235

summary: IL-20 cytokines are involved in the establishment of psoriasis, a common chronic skin inflammation epidemiologically associated with metabolic syndrome, but molecular mechanisms underlying their over-expression remain to be elucidated. We find that keratinocytes (KCs) expressed IL-20 and lymphocytes expressed IL-22 cytokines up-regulation occurs at post-transcriptional level with stabilization of their RNA messengers. Looking at psoriatic epidermis, we observe that the p38/MK2 pathway is not activated but that the RNA-binding protein (RBP) HuR re-localizes in keratinocytes cytoplasm, suggesting post-transcriptional regulation of numerous mRNAs. HuR ribonucleoprotein immunoprecipitations analyzed by high-throughput sequencing (RIP-Seq) identify potential pre-mature and mature RNA targets for uninvolved and involved skin and confirms that HuR activity is displaced from the nucleus to the cytoplasm. Numerous psoriasis up-regulated transcripts are HuR targets and HuR knockdown reduces expression of transcripts like beta-defensin-2, CXCL-10 or IL-2, suggesting an implication of HuR in pathophysiological processes such as morphological, immune and metabolic inflammatory responses. Finally, metabolic disorders affecting psoriatic keratinocytes are responsible for HuR cytoplasmic localization since a decreased activity of the cellular metabolic sensor AMPK, that is observed in human psoriatic epidermis, is sufficient to promote HuR cytosolic localization as well as IL-20 over-production both in human keratinocytes and in vivo in mouse epidermis where it then initiates psoriasis-like histological changes. These results may provide insights into molecular links between metabolism and post-transcriptional networks during chronic inflammation, as illustrated in psoriasis by mechanisms connecting AMPK, HuR and IL-20.

overall_design: Analysis of HuR-binding RNA in uninvolved versus involved psoriatic samples by RIP-Seq. Samples from five different patients were used for both uninvolved and involved skin. RIP-Seq was also made using a control IgG.

first: Genevieve

first: Isabelle

last: Guiraud

first: Matthieu

last: Lacroix

first: Clemence

last: Genthon

first: Stephanie

last: Rialle

first: Jean

last: Joujoux

first: Laurent

last: Meunier

first: Thierry

last: Lavabre-Bertrand

first: Pierre

last: Stoebner

first: Lionel

last: Le Gallic


title: Analysis of bacterial and macrophage transcripts in ISRE-positive and negative infected macrophages

first: Deborah

last: Hung

address: 415 Main Street

department: Infectious Disease Initiative

institute: Broad Institute

summary: To identify bacterial transcripts that may be associated with type I IFN production in Salmonella enterica subsp typhimurium (SL1344) infected macrophages we transformed macrophages with an ISRE-GFP reporter construct and sorted separate populations of GFP positive and GFP negative infected macrophages.  We then did whole transcriptome profiling, collecting both host and bacterial transcripts, for differential expression analysis

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: Analysis of ISRE positive, negative, and mixed populations at two time points (unexposed and 24hours) in duplicate (biological replicates).  A sample consisting of Salmonella prior to infection was also included

first: Roi

last: Avraham

first: Nathan

last: Haseley

first: Douglas

last: Brown

first: Cristina

last: Penaranda

first: Humberto

last: Jijon

first: John

middle: J

last: Trombetta

first: Rahul

last: Satija

first: Alex

last: Shalek

first: Ramnik

last: Xavier

first: Aviv

last: Regev


title: Single cell analysis of macrophages exposed to beads coated with LPS from Salmonella enterica subsp. typhimurium

summary: We present a detailed single cell analysis of the macrophage response to LPS from Salmonella enterica.  By combining single cell transcriptional analysis, fluorescently labeled, LPS-coated beads, and cytometry we are able to distinguish the responses of macrophages that have internalized LPS-coated beads and those that have not.

overall_design: Analysis of 96 single macrophages that were either: left untreated, were exposed to but did not internalize uncoated beads, were exposed to and internalized uncoated beads, were exposed to but did not internalize LPS-coated beads, or were exposed to and did internalize LPS-coated beads.


title: Transcriptome bioinformatical analysis of vertebrate stages of Schistosoma japonicum reveals alternative splicing events

status: Public on Sep 06 2015

city: Shanghai

first: Xinye

last: Wang

state: Shanghai

address: 1239 Siping Road

department: Tongji University School of Medicine

country: China

institute: Institute for Infectious Diseases and Vaccine Development

summary: Alternative splicing is a molecular process that contributes greatly to the diversification of proteome and to gene functions. Understanding the mechanisms of stage-specific alternative splicing can provide a better understanding of the development of eukaryotes and the functions of different genes. Schistosoma japonicum is an infectious blood-dwelling trematode with a complex lifecycle that causes the tropical disease schistosomiasis. In this study, we analyzed the transcriptome of Schistosoma japonicum to discover alternative splicing events in this parasite, by applying RNA-seq to cDNA library of adults and schistosomula. Results were validated by RT-PCR and sequencing. We found 11,623 alternative splicing events among 7,099 protein encoding genes and average proportion of alternative splicing events per gene was 42.14%. We showed that exon skip is the most common type of alternative splicing events as found in high eukaryotes, whereas intron retention is the least common alternative splicing type.

overall_design: BioProject accession number is PRJNA290970, and the SRA Study accession number is SRP061588. Mixed sample of cDNA libaraies of Schistosomula and adult were sequenced using Illumina HiSeq2000.

first: Xindong

last: Xu

first: Xingyu

last: Lu

first: Yuanbin

last: Zhang

first: Weiqing

last: Pan


title: Bulk RNA-seq analysis of the macrophage response to Salmonella enterica subsp. Typhimurium (SL1344) exposure

summary: A time course of the macrophage response to Salmonella exposure analyzing the effects of input cell number as a control for single cell studies

overall_design: Mouse macrophages were exposed to Salmonella enterica for different lengths of time.  Libraries were constructed using either approximately 500,00 macrophages lysed directly on a tissue culture dish (bulk) or using only 150 cells isolated using FACS (sorted).  All libraries were constructed in duplicate (bulk) or triplicate (sorted).  All replicates are biological replicates


title: Transcriptome of honey bee larvae

city: Canberra

first: Sylvain

last: Foret

state: ACT

address: Gould Building #116

department: Research School of Biology

country: Australia

institute: The Australian National University

summary: Honey bee drones, queens and workers have vastly different phenotypes.  Here we profile the the expression level of mRNAs and microRNAs of honeybee, drones, queens and workers at the L5 larval stage (91 hours +/- 1).

overall_design: For both mRNA and miRNA, we analyse five replicates for drones, queens and workers (15 replicates for mRNA and 15 for miRNA).

first: Regan

last: Ashby

first: Ian

last: Searle

first: Ryszard

last: Maleszka


title: Cross-species transcriptomic comparison of in vitro and in vivo mammalian neural cells

status: Public on Sep 05 2015

city: Rochester

first: FENG

last: CUI

state: NY

address: One Lomb Memorial Dr.

department: Thomas H. Gosnell School of Life Sciences

institute: ROCHESTER INSTITUTE OF TECHNOLOGY

summary: We report the first RNA profiing of mammalian neural cells grown in vitro. About 50 million reads are generated by RNA-seq from rat astrocytes, neurons and oligodendrocyte precursor cells in primary culture. These data are compared with theose generated from the correponding mouse neural cells that are acutely purified from brains. Cross-species RNA-seq data analysis revealed hundreds of genes that are differentially expressed between cultured and acutely purified cells. Astrocytes have more such genes compared to neurons and oligodendrocyte precursor cells, indicating that signalling pathways are greatly perturbed in cultured astrocytes.

overall_design: mRNA profiles of rat astrocytes, neurons and oligodendrocyte precursor cells cultured in vitro

first: Peter

last: LoVerso

first: Christopher

last: Wachter

first: Feng

last: Cui


title: APOBEC3B facilitates a functional and necessary link between estrogen receptor mediated transcription and DNA repair

city: London

first: Luca

last: Magnani

address: DuCane road

department: Surgery and Cancer

country: United Kingdom

institute: Imperial College London

summary: Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations.

overall_design: Hormone-depleted MCF-7 breast cancer cell line was treated with estrogen (100 nM), H2O2 (10 mM) or vehicle for 45 minutes. H2AX ChIP-seq was performed using Illumina methodology.

first: Simak

last: Ali

first: Wilbert

last: Zwart


title: An autoregulatory enhancer controls mammary-specific STAT5 functions

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: Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development. We report that the ubiquitously expressed Stat5a/b locus is subject to lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitive sites, H3K27 acetylation and binding by GR and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these STAT5 binding sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that underlies the remarkable abundance of STAT5 and, in turn, controls the efficacy of STAT5-dependent mammary physiology.

overall_design: ChIP-seq for H3K27ac, RNA Pol II, and MED1 in mammary tissues at L1, and ChIP-seq for H3K27ac and GR in mammary tissues at p13. mRNA-seq in WT at L1, line B (GAS2 mutation only) and line C (both GAS1 and GAS2 mutations) at L1 in mammary tissues, and DNase-seq in WT mammary tissues at L1.

first: Gil

last: Metser

first: Ha-Youn

last: Shin

first: Chaochen

first: Kyung-Hyun

last: Yoo

first: Lothar

last: Hennighausen


title: Single cell time course of macrophages exposed to Salmonella enterica subsp. typhimurium

first: Deborah

last: Hung

summary: We present a detailed single cell time course of the macrophage response to Salmonella infection.  By combining phenotypic fluorescent labels with single cell expression analysis we are able to identify gene modules associated with bacterial exposure and bacterial infection.  We also identify other genetic clusters that are expressed heterogenously, ananlyzing both their regulation and their impact on infection

overall_design: Analysis of 192 single cells across 4 time points after Salmonella exposure (MOI 1:1) with one of three different fluorescent labels indicating whether a given cell contained no intracellular bacteria (non-fluorescent), contained dead intracellular bacteria (only pHrodo positive), or contained live intracellular bacteria (pHrodo and GFP positive)

first: Roi

last: Avraham

first: Nathan

last: Haseley

first: Douglas

last: Brown

first: Cristina

last: Penaranda

first: Humberto

last: Jijon

first: John

last: Trombetta

first: Rahul

last: Satija

first: Alex

last: Shalek

first: Ramnik

last: Xavier

first: Aviv

last: Regev


title: Gene expression in livers and thymi of uninjected or MegaFasL-injected C57BL/6 and MSM/Ms mice

status: Public on Sep 04 2015

city: Boston

first: Alexander

last: Poltorak

address: 150 Harrison avenue

institute: Tufts University

summary: Here we examine the expression of genes in unstimulated or following intraperitoneal injection with MegaFasL in MSM/Ms and C57BL/6 mouse livers and thymi

sample_organism:
Mus musculus domesticus
Mus musculus molossinus

overall_design: This dataset is from two groups of 2 mice (1 MSM/Ms [MSM] and 1 C57BL/6 [B6] mouse) - either untreated (#1-#4) or injected intraperitoneally with 1.5ug of MegaFasL (#5-#8). Each group has four (4) samples - one liver (L) and one thymus (T) sample from each mouse. The liver and thymi were harvested from the same mouse. Unstimulated samples: 1_S1 = B6 T, 2_S=MSM T, 3_S3=B6 L, 4_S4=MSM L; MegaFasL-injected samples: 5_S1=B6 T, 6_S2=MSM T, 7_S3=B6 L, 8_S4=MSM L.

first: Daniel

last: Ram


title: Transition from somatic embryo to friable embryogenic callus in cassava: Dynamic changes in cellular structure, physiological status, and gene expression profiles

first: Qiuxiang

last: Ma

address: 300 Fenglin Road

institute: Institute of Plant Physiology and Ecology, SIBS,CAS

summary: Friable embryogenic callus (FEC) has been considered as the most suitable materials for efficient genetic transformation of cassava. Due to heavy genotype dependence on FEC induction and being amenable to somaclonal variation, the production and maintaining of reliable FEC is a limitation factor for cassava genetic transformation. Identification of key elements involving in biological processes from somatic embryos (SEs) to the FEC at different stages will provide critical insights for FEC improvement. Cytological observation showed the dramatic change of subcellular structures among SEs, fresh FEC (FFEC) and old FEC (OFEC). Decrease of sucrose and increase of fructose and glucose were detected in OFEC. A total of 6871 differentially expressed genes (DEGs) were identified from SEs, FFEC and OFEC by RNA-seq. Analysis of the DEGs showed that FEC induction was accompanied by the process of dedifferentiation, whereas the epigenetics modification occurred during the continuous subculturing process. The cell structure was reconstructed, mainly including the GO terms of "cell periphery" and "external encapsulating structure"; in parallel, the internal mechanisms were changed correspondingly, including the metabolisms of process (glycolysis) and compounds(alanine, aspartate, and glutamate). The significant reduction of genomic DNA methylation in OFEC indicates altered gene expression via chromatin modification. These results indicate that the induction and long-term subculture of FEC is a complicated biological process involving changes of genome modification, gene expression and subcellular reconstruction. The findings will be useful for improving FEC induction and maintenance from farmer-preferred cassava cultivars that are recalcitrant to genetic transformation, hence improving cassava through genetic engineering.

overall_design: Two-week-old SEs cultured on GD, FFEC emerging from SEs, and 9-month-old OFEC were used for analysis. Each sample was mixtured from six indepedent replicates and were collected for RNA extraction respectively.

first: Jun

last: Yang

first: Peng

last: Zhang


title: Dosage effect on small RNA transcripts in maize copy number alterations

city: Ames

first: Thomas

last: Peterson

state: IA

address: 2288 Molecular Biology Building

department: Genetics, Development and Cell Biology

institute: Iowa state University

summary: We sequenced 10 small RNA samples. 6 samples were taken from the 14-day old maize seedling tissue, and the other 4 samples were taken from 14-day old maize root tissue.

overall_design: Examination of small RNA levels in each individual sample

first: Tao

last: Zuo

first: Dafang

last: Wang


title: Enrichment of H3K9me2 on unsynapsed chromatin in C. elegans does not target de novo sites [ChIP-Seq]

city: Syracuse

first: Eleanor

last: Maine

state: New York

address: 107 College Pl

department: Biology

institute: Syracuse University

summary: Characterization of the H3K9me2 distribution in the C. elegans adult hermaphrodite genome using ChIP-Seq

overall_design: Examination of H3K9me2 in two different nematode strains: fer-1 control and fer-1;him-8 mutant, where the X chromosomes are unsynapsed.  The fer-1 mutation prevents production of embryos.

first: Yiqing

last: Guo


title: Photophysiological and Genetic Factors Underlying Fast Photosynthetic Growth of Unicellular Cyanobacterium Synechococcus sp. PCC 7002

first: Ryan

last: McClure

address: 650 Albany St.

institute: Boston University

summary: Application of genome-scale 'omics approaches to dissect subcellular pathways and regulatory networks governing the fast-growing response of Synechococcus sp. PCC 7002 response to variable irradience levels.

overall_design: We employed controlled cultivation and next-generation sequencing technology to identify transcriptional responses of euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 grown under steady state conditions at six irradiance levels ranging from 33 to 760 µmol photons m-2 sec-1.

first: Hans

middle: C

last: Bernstein

middle: S

first: Eric

middle: A

last: Hill

first: Lye

last: Markillie

first: William

last: Chrisler

first: Margie

last: Romine

first: Matthew

last: Posewitz

first: Donald

last: Bryant

first: Allan

last: Konopka

first: James

last: Fredrickson

last: Beliaev


title: The Polycomb protein Pcgf2/Mel18 regulates mesoderm cell fate-specification of embryonic stem cells through multiple mechanisms

city: Barcelona

first: Enrique

last: Blanco

address: Dr. Aiguader 88

department: Gene Regulation, Stem Cells and Cancer

institute: Center for Genomic Regulation (CRG)

summary: Polycomb complexes are essential regulators of stem cell identity, yet very little is known about their molecular mechanisms during cell differentiation. Pcgf proteins (Pcgf1/2/3/4/5/6) are core subunits of the Polycomb repressive complex 1 (PRC1). It has been recently proposed that specific Pcgf proteins are associated to particular PRC1 complexes, yet the molecular and biological functions of different Pcgf proteins remains largely elusive. Using specific differentiation protocols, we have elucidated a role for Pcgf2/Mel18 in specifically regulating mesoderm differentiation. Mechanistically, during early cardiac mesoderm differentiation, Pcgf2/Mel18 functions as a classical Polycomb protein by repressing pluripotency, lineage specification, late cardiac differentiation and negative regulators of the BMP pathway, yet Pcgf2/Mel18 also positively regulates the expression of key mesoderm transcription factors, revealing a novel function of Pcgf2/Mel18 in gene activation during cardiac differentiation. Mel18 depletion results in an unbalance of pathways that positively and negatively regulate cardiac differentiation. We propose that Mel18 is a novel epigenetic factor that controls mesoderm differentiation by opposing molecular mechanisms.

overall_design: List of ChIPseq samples: Mel18 in ESCs and MES, Ring1b, RYBP and Cbx2 in MES, IgG in MESs. List of RNAseq experiments: Mel18 KD, Ring1b KO and CTR in ESCs, Mel18 KD and CTR in MES, Mel18 KD and CTR in CMs.

first: Lluís

last: Morey

first: Alexandra

last: Santanach

first: Luigi

last: Aloia

first: Elphege

last: Nora

first: Benoit

last: Bruneau

first: Luciano

last: DiCroce


title: Dosage effect on genes in maize copy-number alterations

first: Thomas

last: Peterson

summary: We sequenced 9 mRNA samples taken from the 14-day old maize seedling tissue. These 9 samples are comprised by 3 genotypes and 3 replications.

overall_design: Examination of mRNA levels in each individual genotype.

first: Tao

last: Zuo


title: Cellular response to proteasome inhibition by Bortezomib

city: Stanford

last: Wandless

state: California

address: 318 Campus Dr. W350 Clark Ce

department: Chemical and Systems Biology

institute: Stanford University

summary: Analysis of the coordinated transcriptional reponse to proteasome inhibition

overall_design: mRNA profiles of NIH3T3 cells which stably expressing DD-sfGFP were generated by deep sequencing using Illumina HiSeq. The samples were collected from indicated timepoints after exposed to proteasome inhibition by Bortezomib.

first: Yusuke

last: Miyazaki


title: Enrichment of H3K9me2 on unsynapsed chromatin in C. elegans does not target de novo sites [RNA-Seq]

first: Eleanor

last: Maine

summary: We sequenced mRNA isolated from adult hermaphrodite met-2;him-8 and him-8 gonads in order to determine the gene expression profile of germ cells with or without H3K9me2.

overall_design: Examination of mRNA profiles in nematode gonad of met-2;him-8 (without H3K9me2) and him-8 (with H3K9me2)

first: Yiqing

last: Guo


title: Enrichment of H3K9me2 on unsynapsed chromatin in C. elegans does not target de novo sites

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

overall_design: Refer to individual Series


title: Transcriptomic analysis of cultured corneal endothelial cells as a validation for their use in cell-replacement therapy

first: Anthony

middle: J.

last: Aldave

address: 200 Stein Plaza DSERC 3-143

department: Ophthalmology

institute: Stein Eye Institute, UCLA

summary: The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelium (evHCEnC) with that of primary HCEnC and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level primary HCEnC most closely resemble evHCEC and thus represent a viable therapeutic option for managing corneal endothelial dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of the potential clinical utility of the cultured HCEnC for the management of corneal endothelial cell failure.

overall_design: Transcriptomes from ex vivo corneal endothelium, primary cultures and three cell lines were compared. Three samples of each endothelial cell group were submitted for RNA sequencing for a total of 15 samples. The transcriptome for the ex vivo corneal endothelium was used as the reference (i.e., proxy for in vivo corneal endothelium). Transcript abundances for a subset of genes associated with corneal endothelial cell function or disease were validated with qPCR and western blot. Samples of ex vivo endothelium used for validation were independent replicates not used for RNA-sequencing.

first: Ricardo

last: Frausto

first: Derek

last: Le


title: RNA-seq analysis of differential gene expression in muscle from Holstein Friesian bulls undergoing diet restriction and compensatory growth

city: Co. Meath

first: Kate

last: Keogh

address: Dunsany

department: Animal and Bioscience Department

institute: Teagasc Grange

summary: The objective of this study was to examine changes in muscle gene expression of growing bulls during a period of dietary energy restriction followed by a period of subsequent realimentation and compensatory growth. Purebred Holstein Friesian bulls (n=20) were assigned to one of two feeding treatments (i) restricted feed allowance for 125 days (n=10) followed by ad libitum access to feed for a further 55 days or (ii) a control group with ad libitum access to feed through out the 180 days trial (n=10).  The first 125 days of the trial were denoted as Peirod 1, during which treatment groups were fed differentially. The subsequent 55 days, denoted as Period 2 during which all bulls were fed ad libitum. All bulls received the same diet of 70% concentrate 30% grass silage through out the experimental trial,with the amount of feed provided different dependnet on each treatment group. Muscle biopsies were collected at 2 time points (end of the differential feeding in Period 1 (d 120) and during the realimentation phase in Period 2 (d 15 of re-alimentation). RNA was extracted and muscle gene expression was examined using RNAseq technology and bioinformatic analysis. During the differential feeding period, over-represented pathways including fatty acid beta oxidaiton, oxidative phosphorylation and TCA cyclewere identified, which indicate utilisation of lipid sotes for energy utilisaiton and also alterations in energy produciton during dietary restriciton in muscle. During the realimentation period, pathways involved in energy produciton were over-represented, with the direction of fold changes opposite to that of these pathways in Period 1. additionally, a number of genes involved in cell division and cellular proliferation were up-regulated during compensatory growth in re-alimentation, thereby promoting accelerated cell growth and proliferation in muscle tissue of animals experiencing compensatory growth. This information can be exploited in genomic breeding programmes to assist selection of cattle with a greater ability to compensate following a period dietary restriction.

overall_design: 40 muscle RNA samples were analysed in total. 10 samples were from muscle biopsies collected at the end of a period of dietary restriction (d 120) and 10 samples were from muscle biopsis collected during the initial stages of compensatory growth (d 15 of re-alimentation). In addition,  RNA was also anlaysed from 10 samples collected from animals fed a libitum at each of these two timepoints.

first: David

last: Kenny

first: Alan

last: Kelly

first: Paul

last: Cormican

first: Matthew

last: McCabe

first: Sinead

last: Waters


title: miR-431 promotes differentiation and regeneration of old skeletal muscle by targeting Smad4

city: Daejeon

first: sujin

last: Baek

address: Gwahank-ro 125

department: MGRC

institute: KRIBB

summary: we identified novel age-related microRNAs through comparative analysis of miRNA expression profiles in young and old myoblasts using next generation sequencing (NGS)

overall_design: small RNA profiles of young mouse and old mouse were generated by deep sequencing, in each four samples, using Illumina GAIIx

first: Kwang-Pyo

last: Lee

first: Su-Jin

first: Ki-Sun

last: Kwon

first: Seon-Young

last: Kim


title: Cellular response to unfolded proteins in cytosol and nucleus

first: Thomas

last: Wandless

summary: Analysis of the coordinated transcriptional response to unfolded proteins in cytosol and nucleus utilizing mRNA-seq

overall_design: mRNA profiles of NIH3T3 cells which stably expressing DD-sfGFP were generated by deep sequencing using Illumina HiSeq2000. The samples were collected from indicated timepoints after Shield-1 removal.

first: Yusuke


title: RNAseq and Lariat sequencing of different S. pombe growth conditions

status: Public on Sep 03 2015

city: Ithaca

first: Jeffrey

last: Pleiss

address: 451 Biotech

department: Molecular Biology and Genetics

institute: Cornell University

summary: We performed lariat sequencing to profile the diversity of spliced RNA lariats in S. pombe identify annotated and alternate introns.

overall_design: Three different growth conditions were used to grow S. pombe wt and S. pombe Δdbr1. Lariat sequencing of the Δdbr1 strains and RNAseq of the wt and Δdbr1 strains were done to profile intron lariats and exon-exon junctions in RNA transcripts.

first: Nick

last: Stepankiw

first: Elizabeth

last: Fogarty

first: Andrew

last: Grimson


title: Next Generation Sequencing Identifies Differentially Expressed Genes in Zebrafish Embryos and Larvae Following Benzo[a]pyrene Exposure

city: Gainesville

first: Xiefan

last: Fang

state: Florida

address: 1200 Newell Drive, ARB R1-148

department: Pediatrics

institute: University of Florida

summary:
Purpose: This study aimed to identify differentially expressed genes and transcripts in zebrafish embryos and larvae following benzo[a]pyrene (BaP) exposure.
Methods: Adult zebrafish (2 males × 4 females, N=6 replicate tanks for each treatment) were acclimated for 7 days in an 818 Low Temp Illuminated Incubator (Precision Scientific, Chennai, India) at 28.5°C. Next, adult fish were waterborne exposed to control or 50 μg/L (ppb) BaP for 7 days; ethanol was used as vehicle solvent, and final ethanol concentration was 0.1 mL/L (100 ppm) in all treatment groups. This dose of ethanol is not teratogenic to zebrafish. Water was changed and/or re-dosed daily. From day 7 to 11 of the parental exposure, eggs were collected, counted, and raised in normal conditions (control) or continuously exposed to 50 μg/L BaP until 3.3 and 96 hours post fertilization (hpf). At 3.3 or 96 hpf, embryos (200/pool) or larvae (10/pool) were collected and pooled. Total RNA was isolated for transcriptomic RNA sequencing with Illumina HiSeq2000 (2X100bp). RNA-seq reads were uploaded to the galaxy platform https://main.g2.bx.psu.edu/. RNA-seq reads were trimmed, filtered, and aligned to the zebrafish genome (Danio_rerio.Zv9.68) with the Tophat for Illumina tool. Counting and annotation of RNA-seq reads were performed with Partek Genomics Suite version 6.11. Refseq Transcripts (2013-04-10) and Ensembl Transcripts release 70 databases were used for gene and transcript annotation. Differential expression of gene and transcript reads between treatments was analyzed with R package EdgeR. Genes/transcripts with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Differentially expressed genes were defined as genes with altered expression at either gene or transcript level.
Results: Differential expression analysis with EdgeR revealed that gene expression was vastly different between 3.3 hpf zebrafish embryos and 96 hpf larvae. Using Refseq annotation, we found that 10644 out of 13950 transcribed zebrafish genes were differentially expressed between the two developmental time-points, with 5961 up-regulated genes and 4683 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. Similarly, using Ensembl annotation, 16529 out of 19886 transcribed zebrafish genes were differentially expressed, with 9318 up-regulated genes and 7211 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. In 3.3 hpf embryos, four genes and seven transcripts were differentially expressed after BaP exposure. In 96 hpf larvae, 447 and 484 zebrafish genes were significantly up- and down-regulated, respectively, by BaP exposure.
Conclusions: Parental and developmental BaP exposure caused gene expression changes in zebrafish embryos and larvae.

overall_design: Illumina HiSeq2000 deep sequencing was used to generate transcriptomic profiles for BaP-exposed 3.3 hpf zebrafish embryos (n=3 for control, n=3 for BaP) and 96 hpf larvae (n=2 for control, n=2 for BaP).

first: Jones

last: Corrales

first: Cammi

last: Thornton

first: Mary

last: Duke

first: Brian

last: Scheffler

first: Kristine

last: Willett


title: The ability of PML/RARα to initiate leukemia is associated with markedly increased proliferation of promyelocytes despite minor changes in the transcriptome and epigenome.

city: Miami

first: Maria

middle: Eugenia

last: Figueroa

state: FL

address: 1501 NW 10th Ave, BRB 709A, Locator code C227

department: Human Genetics

institute: University of Miiami

summary: Acute Promyelocytic Leukemia is characterized by the accumulation in the blood and bone marrow of promyelocytes. The PML/RARα fusion protein is identified as the primary abnormality implicated in the pathology, and is believed to prevent transcription of genes necessary for normal myeloid development and differentiation. Identifying its targets is critical to comprehend the road to pathogenesis. To understand how PML/RARα, in the absence of secondary lesions, alters gene expression, DNA methylation and proliferation we used a novel experimental and sorting strategy to study normal versus preleukemic promyelocytes in vivo. Expression and methylation profiling analyses were performed on highly purified samples. Surprisingly, despite its ability to initiate leukemia, PML/RARα had overall minor effects on both the transcriptome and epigenome. Important regulators of the myeloid maturation program were not altered but, remarkably, PML/RARα promyelocytes showed strong downregulation of secondary and tertiary granule genes. Subtle changes were also observed on the DNA methylation profile, with PML/RARα predominantly mediating hypomethylation. We performed intersection studies between altered loci and previously described PML/RARα binding sites but found little overlap. Importantly, we show for the first time that PML/RARα on its own increases proliferation, and that this increased proliferation correlates with the ability to initiate leukemia.

overall_design: DNA methylation profiling in CD34(+) early promyelocytes and CD34(-) late promyelocytes cells from PML-RARa transgenic mice vs. control mice.

first: Coline

last: Gaillard

first: Scott

last: Kogan


title: Cellular response to heat shock and ER stress

summary: Analysis of the coordinated transcriptional reponse to heat shock and ER stress

overall_design: mRNA profiles of NIH3T3 cells which stably expressing DD-sfGFP were generated by deep sequencing, in triplicate, using Illumina HiSeq. The samples were collected from indicated timepoints after exposed to either heat stress or ER stress.

last: Miyazaki


title: Pre-patterning of differentiation-driven nuclear lamin A/C-associated chromatin domains by GlcNAcylated histone H2B

city: Oslo

first: Philippe

last: Collas

address: PO Box 1112 Blindern

department: Institute of Basic Medical Sciences

country: Norway

institute: University of Oslo

summary: Dynamic interactions of nuclear lamins with chromatin through so-called lamin-associated domains (LADs) contribute to spatial arrangements of the genome. Here, we provide evidence for pre-patterning of differentiation-driven formation of lamin A/C LADs by domains of histone H2B modified by the nutrient sensor O-linked N-acetylglucosamine (H2BGlcNAc), which we term GADs. We demonstrate a two-step process of lamin A/C LAD formation during in vitro adipogenesis, involving (i) a spreading of lamin A/C-chromatin interactions during the transition from progenitor cell proliferation to cell cycle arrest, and (ii) a genome-scale redistribution these interactions through a process of LAD ‘exchange’ within hours of adipogenic induction. Chromatin state modeling reveals that lamin A/C LADs can be found both in active and repressive chromatin contexts which can be influenced by cell differentiation status. De novo formation of adipogenic lamin A/C LADs occurs non-randomly on GADs, consisting of megabase-size intergenic and repressive chromatin domains. Accordingly, while pre-differentiation lamin A/C LADs are gene-rich, post-differentiation LADs harbor repressive features reminiscent of lamin B1 LADs. Moreover, release of lamin A/C from genes directly involved in glycolysis concurs with their transcriptional upregulation after adipogenic induction, and with concordant downstream elevations in H2BGlcNAc levels and O-GlcNAc cycling. Our results unveil an epigenetic pre-patterning of adipogenic LADs by GADs, suggesting a coupling of developmentally regulated lamin A/C-genome interactions to a metabolically sensitive chromatin modification.

overall_design: Examination of LMNA and H2BGlcNAc binding in ASCs across differentiation

first: Akshay

last: Shah

first: Torunn

last: Rønningen

first: Anja

last: Oldenburg

first: Erwan

last: Delbarre

first: Jan

middle: O

last: Moskaug


title: ERRγ links adaptive neuronal metabolism to spatial learning and memory [ChIP-Seq]

city: La Jolla

first: Ruth

last: Yu

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute

summary: Neurons utilize glucose to generate adenosine triphosphate (ATP) essential for their survival, excitability and synaptic signaling, as well as initiating changes in neuronal structure and function. Defects in oxidative metabolism and mitochondria functions are also associated with aging and diverse human neurological diseases1-4. While neurons are known to adapt their metabolism to meet the increased energy demands of complex behaviors such as learning and memory, the molecular underpinnings regulating this process remain poorly understood4-6. Here we show that the orphan nuclear receptor estrogen related receptor gamma (ERRγ) becomes highly expressed during retinoic-acid induced neurogenesis and is widely expressed in neuronal nuclei throughout the brain. Mechanistically, we show that ERRγ directly orchestrates the expression of networks of genes involved in mitochondrial oxidative phosphorylation and energy generation in neurons. The importance of this regulation is evidenced by decreased adaptive metabolic capacity in cultured neurons lacking ERRγ, and reduced long-term potentiation (LTP) in ERRγ-/- hippocampal slices. Notably, the defect in LTP was rescued by the metabolic intermediate pyruvate, functionally linking the ERRγ knockout metabolic phenotype and memory formation. Consistent with this notion, mice lacking neuronal ERRγ exhibit defects in spatial learning and memory. These findings implicate ERRγ in the metabolic adaptations required for long-term memory formation.

overall_design: We used ChIP-Seq analysis to determine the genome-wide binding of ERRγ in neurons derived from ES cells.

first: Liming

last: Pei

first: Ronald

middle: M

last: Evans


title: Tracking distinct RNA populations using efficient and reversible covalent chemistry

first: Erin

middle: Elizabeth

last: Duffy

address: 200 Longwood Ave, Goldenson 405

department: Neurobiology

institute: Harvard Medical School

summary: We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.

overall_design: s4U metabolic labeling of RNA in 293T cells, followed by biochemical enrichment of labeled RNA with two biotinylation reagents, RNAs >200nt and miRNAs in separate experiments

first: Michael

last: Rutenberg-Shoenberg

first: Catherine

middle: D

last: Stark

first: Robert

middle: R

last: Kitchen

first: Mark

last: Gerstein

first: Matthew

last: Simon


title: Sensing Cardiac Electrical Activity with a Cardiomyocyte Targeted Optogenetic Voltage Indicator

status: Public on Sep 02 2015

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

institute: Universitaetsmedizin Goettingen

summary: Rationale: Monitoring and controlling cardiomyocyte activity with optogenetic tools offers exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level.   Objective: To test the hypothesis that cardiomyocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electrical activity in isolated cardiomyocytes and the whole heart as well as function and maturity in induced pluripotent stem cell (iPSC)-derived cardiomyocytes.   Methods and Results: We first generated mice with cardiomyocyte-restricted expression of VSFP2.3 and demonstrated distinct sarcolemmal localization of VSFP2.3 without any signs for associated pathologies (assessed by echocardiography). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch-clamping. The utility of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiomyocytes. Optical cardiograms (OCGs) could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in iPSC-derived cardiomyocytes.  Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof-of-concept for its utility in optogenetic sensing of physiological and pathological excitation in mature and immature cardiomyocytes in vitro and in vivo.

overall_design: Determination of transgene (VSFP2.3) cardiotoxicity

first: Wolfram-Hubertus

last: Zimmermann


title: Cell type transcriptomics of hypothalamic energy-sensing neuron responses to fasting

city: Ashburn

first: Ken

last: Sugino

state: VA

address: 19700 Helix Dr

institute: Janelia Research Campus

summary: AGRP neurons are a hypothalamic population that senses physiological energy deficit and consequently increases appetite. Molecular and cellular processes for energy-sensing and elevated neuronal output are critical for understanding the central nervous system response to energy deficit states, such as during weight-loss. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss but, in adult mice, this has been limited by technical challenges. We report high-quality gene expression profiles of AGRP neurons under well-fed and energy deficit states. For comparison, we also analyzed POMC neurons, an intermingled population that suppresses appetite. This data newly identifies cell type-selective involvement of signaling pathways, ion channels, neuropeptides, and G-protein coupled receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders.

overall_design: Examination of 2 different neuronal cell types under 2 conditions.

first: Trace

last: Henry

first: Scott

last: Sternson


title: Expression profiling of DT40 chicken B cell line by RNA-seq

city: Bethesda

first: Andrew

last: Oler

state: MD

address: 31 Center Drive, Room 3B62E

department: Bioinformatics and Computational Biosciences Branch (BCBB)

institute: NIAID/NIH

summary: A single replicate of exponentially growing DT40 CL18 chicken B lymphoma cells were harvested and extracted RNA was subjected to Illumina GAIIx paired-end sequencing to determine global gene expression.

overall_design: Single replicate RNA-seq expression analysis of DT40 cells.

first: Julien

last: Häsler

first: Stefan

last: Muljo


title: Chromatin interaction analysis reveals changes in small chromosome and telomere clustering between epithelial and breast cancer cells

city: Durham

first: Rasim

last: Barutcu

state: NC

address: 6 Davis Drive

institute: ScitoVation

summary:
Background:
Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines.

Results:
Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways related to repression of WNT signalling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells.

Conclusions:
We show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.

overall_design:
Hi-C experiments were conducted in MCF-7 and MCF-10A parental cells using the HindIII restriction enzyme.

The RNA-seq data associated with this study is deposited under the GEO accession number GSE71862.

first: Ahmet

first: Bryan

last: Lajoie

first: Rachel

middle: P

last: McCord

first: Coralee

middle: E

last: Tye

first: Deli

last: Hong

first: Terri

last: Messier

first: Gillian

last: Browne

first: Andre

last: van wijnen

first: Jane

last: Lian

first: Janet

last: Stein

first: Job

last: Dekker

first: AnthonyTerri

middle: N

last: Imbalzano

first: Gary


title: Transcriptional variation associated with cactus host plant adaptation in Drosophila mettleri populations

city: Colorado Springs

first: Jeremy

last: Bono

state: CO

address: 1420 Austin Bluffs Parkway

institute: University of Colorado Colorado Springs

summary: Although the importance of host plant chemistry in plant-insect interactions is widely accepted, the genetic basis of adaptation to host plants is poorly understood. Here, we investigate transcriptional changes associated with a host plant shift in Drosophila mettleri. While D. mettleri is distributed mainly throughout the Sonoran Desert where it specializes on columnar cacti (Carnegiea gigantea and Pachycereus pringleii), a population on Santa Catalina Island has shifted to coastal prickly pear cactus (Opuntia littoralis). We compared gene expression of larvae from the Sonoran Desert and Santa Catalina Island when reared on saguaro (C. gigantea), coastal prickly pear, and laboratory food. Consistent with expectations based on the complexity and toxicity of cactus relative to laboratory food, within population comparisons between larvae reared on these food sources revealed transcriptional differences in detoxification and other metabolic pathways. The majority of transcriptional differences between populations on the cactus hosts were independent of the rearing environment, and included a disproportionate number of genes involved in processes relevant to host plant adaptation (e.g. detoxification, central metabolism, and chemosensory pathways). Comparisons of transcriptional reaction norms between the two populations revealed extensive shared plasticity that likely allowed colonization of coastal prickly pear on Santa Catalina Island. We also found that while plasticity may have facilitated subsequent adaptive divergence in gene expression between populations, the majority of genes that differed in expression on the novel host were not transcriptionally plastic in the presumed ancestral state.

overall_design:
mRNA profiles of third instar larvae from two different populations reared on three food types was sequenced on two lanes of an Illumina HiSeq 2000

Please note that the de novo assembly gives names to transcripts with the following convention: compXXX_cX_seqX.  The first two identifiers (compXX_cX) are equivalent to a  gene  while the  'seq' identifier might refer to different isoforms or splice variants, etc.  Therefore, for example, a gene might be comp123_c0, and this could have multiple sequences corresponding to different isoforms or splice variants.  Since the analysis was carried out at the gene level, the program internally merged the multiple sequences together for each gene to generate the count matrix (AllGenesint.counts.matrix.txt) (i.e. it only includes comp123_c0),  while the file from the assembly (i.e. Trinity.fasta) also include the individual sequences with the  'seq' identifier.

first: Kim

last: Hoang

first: Luciano

last: Matzkin


city: Guangzhou

first: Rui

last: Xia

state: Guangdong

address: 483 Wushan Rd, Tianhe

department: Horticulture

institute: South China Agricultural University

summary: We constructed four PARE libraries for the identification of miRNA target genes in Norway spruce.

overall_design: PARE libraries were constructed from four different tissues

first: Jing

last: Xu

first: Blake

last: Meyers


title: Distinct EMT programs control normal stem cells and cancer stem cells of mammary tissue

city: Cambridge

first: Xin

last: Ye

state: MA

address: 9 Cambridge Center

department: Whitehead Institute

institute: Whitehead Institute

summary: Cancer stem cells (CSCs) are proposed to be responsible for metastatic dissemination and clinical relapse in a variety of cancers. Analogies between CSCs and normal tissue stem cells (SC) has led to the notion that CSCs often co-opt the normal SC program of their tissue-of-origin. The cell-biological program termed epithelial-mesenchymal transition (EMT) has been found to encourage entrance of normal and neoplastic mammary cells into the corresponding SC states. Using genetically engineered knock-in reporter mouse lines, we demonstrate that in the murine mammary lineage, the paralogous EMT-inducing transcription factors Snail and Slug, are selectively exploited by CSCs and normal SCs respectively. Slug, when expressed at physiological levels, only activates a partial EMT program and is dispensable in CSCs. In contrast, Snail drives a far more complete transition into the mesenchymal state and controls both tumor-initiation and metastatic dissemination. Consistent with their functional distinctions, Snail controls far more target genes than Slug, and their distinct functions are determined by their divergent N-terminal domains. Our findings underscore fundamental distinctions between the SC program operating in normal and neoplastic SCs, and hint for potential avenues of selective therapeutic elimination of breast CSCs.

overall_design: We sought to understand differential ability to activate the EMT program in breast cancer cells by transcription factors Snail and Slug. Hence, we mapped genome-wide Snail and Slug binding sites in murine MMTV-PyMT breast cancer cell lines that express high level of Snail or high level of Slug respectively. Specifically, we performed Snail ChIP seq in the mesenchymal pBl.3G cells, and Slug ChIP-seq in the epithelial pBl.1G cells.

first: Wai

middle: L

last: Tam

first: Robert

last: Weinberg


title: Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage

city: Bar Harbor

first: Petko

last: Petkov

state: Maine

address: 600 Main Street

department: Research

institute: The Jackson Laboratory

summary: We report a novel technique, Affinity-seq, that for the first time identifies both the genome-wide binding sites of DNA-binding proteins and quantitates their relative affinities. We have applied this in vitro technique to PRDM9, the zinc-finger protein that activates genetic recombination, obtaining new information on the regulation of hotspots, whose locations and activities determine the recombination landscape. We identified 31,770 binding sites in the mouse genome for the PRDM9Dom2 variant. Comparing these results with hotspot usage in vivo, we find that less than half of potential PRDM9 binding sites are utilized in vivo. We show that hotspot usage is increased in actively transcribed genes and decreased in genomic regions containing H3K9me2/3 histone marks or bound to the nuclear lamina. These results show that a major factor determining whether a binding site will become an active hotspot and what its activity will be are constraints imposed by prior chromatin modifications on the ability of PRDM9 to bind to DNA in vivo. These constraints lead to the presence of long genomic regions depleted of recombination.

overall_design: The terminal zinc finger domain of PRDM9Dom2 (PRDM9ΔZnF1Dom2, 412–847 aa), the allele present in C57BL/6J (B6) mice was cloned and tagged with 6His-HALO and then expressed in E. coli. DNA sheared to 180–200 bp is provided in considerable excess to provide competition between DNA binding sites. Following binding, DNA–protein complexes are then isolated on streptavidin beads and the DNA extracted for deep sequencing. Two replicate Affinity-seq samples were sequenced at 100-bp reads using the Illumina HiSeq 2500. Alignments to the mm9 mouse genome were obtained utilizing BWA v1.2.3 with default parameters and reads which failed to align to unique positions in the genome were discarded. Peaks were called individually for the two replicates with MACS2 at a p value threshold of 0.01 utilizing a control dataset obtained by sequencing the input DNA and subsequently compared, leading ultimately to combining the two replicates for definitive analysis.

first: Michael

last: Walker

first: Kenneth

last: Paigen