title: The photoreceptor chaperone FHY1 has an independent role in gene modulation and plant development under far-red light [RNA-Seq]

first: Bosheng

last: Li

summary: To incorporate the far-red light (FR) signal into a strategy for optimizing plant growth, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) mediates the nuclear translocation of the FR photoreceptor phytochrome A (phyA) and facilitates the association of phyA with the promoters of numerous associated genes crucial for the response to environmental stimuli. However, whether FHY1 plays additional roles following FR irradiation remains elusive. Here, by the global identification of FHY1 chromatin association sites through ChIP-seq analysis and by the comparison of FHY1-associated sites with phyA- associated sites, we demonstrated that nuclear FHY1 can either act independently of phyA or act in association with phyA to activate the expression of distinct  target genes. We also determined that phyA can act independently of FHY1 in regulating phyA-specific target genes. Furthermore, we determined that the independent FHY1 nuclear pathway is involved in crucial developmental aspects, as in the case of inhibited seed germination under FR during salt-stress. Notably, the differential presence of cis-elements and transcription factors in common and unique FHY1 and/or phyA associated genes are indicative of the complexity of the independent and coordinated FHY1 and phyA pathways. Our study uncovers new aspects of FHY1 function beyond its currently recognized role in phyA-dependent photomorphogenesis

overall_design: The 35S: GFP-FHY1 fhy1-1 transgenic line and the fhy1-1 mutant were grown under the same light conditions used  (D4d+FR3h) for RNA preparation and sequencing. Three biologically replicates were subjected to high-throughput Solexa (Illumina) sequencing.

first: Fang

last: Chen

first: Jordan

last: Demone

first: Jean-Benoit

last: Charron

first: Xiarong

last: Shi

first: Xing

middle: W

last: Deng


title: RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor

status: Public on Aug 04 2014

city: Bryn Mawr

first: Joshua

middle: A

last: Shapiro

state: PA

address: 101 N Merion Ave

department: Biology

country: USA

institute: Bryn Mawr Collete

type: Expression profiling by high throughput sequencing

summary: We report 6 genes that were found to be regulated by SoxR in stationary phase when the bacteria produce the antibiotic actinorhodin.  Five of these genes are previously confirmed SoxR targets; the sixth is a novel SoxR-target.

overall_design: RNA was obtained from 1-day or 3-day old wild type or soxR null mutant strains grown on R2YE agar complex medium. Biological replicates were obtained for 3 day-old samples. RNA was sequenced in an Illumina HiSeq 2000 sequencer for single reads of 100 bp and mapped to the S. coelicolor M145 genome.

first: Monica

last: Chander

first: Nawar

last: Naseer


title: Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

city: Nijmegen

first: Gert Jan

last: Veenstra

address: Geert Grooteplein 28

department: Molecular Developmental Biology

country: Netherlands

institute: Radboud University

summary: Recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells. In contrast, single cell proteomics analyses have been restricted to targeted analyses, for example using flow cytometry with GFP fusions or mass cytometry. Here, we performed global absolute protein quantification of single Xenopus laevis eggs using mass  spectrometry-based proteomics. We quantified over 5800 proteins, thus representing the largest single cell proteome that has been characterized to date. Absolute protein amounts in single eggs are highly comparable, thus indicating a tight regulation of global protein abundance. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identified 439 proteins that significantly change in abundance during early embryogenesis. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in Xenopus leavis eggs.

overall_design: RNA-seq in Xenopus laevis of 5 replicates of both single eggs and single embryos.

first: Arne

middle: H

last: Smits

first: Rik

middle: G

last: Lindeboom

first: Matteo

last: Perino

first: Simon

middle: J

last: van Heeringen

first: GertJan

middle: C

first: Michiel

last: Vermeulen


title: Critical role of transient activation of human endogenous retroviruses during reprogramming toward pluripotency

city: Kyoto

first: Akira

last: Watanabe

state: Kyoto

address: Rm504, Miyako Bldg, 233, Isa-cho, kamigyo-ku

country: Japan

institute: CyberomiX Inc

type: Genome binding/occupancy profiling by high throughput sequencing

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: Critical role of transient activation of human endogenous retroviruses during reprogramming toward pluripotency (Chip-Seq)

summary: We recently showed that some human induced pluripotent stem cell (iPSC) clones were defective in neural differentiation and were marked with the activation of long term repeats (LTRs) of human endogenous retroviruses (HERVs). We herein demonstrated that these LTRs were transiently overexpressed during the generation of iPSCs and contributed to reprogramming. When the generation of iPSCs was completed, LTRs were re-suppressed to levels similar to those in human ES cells. However, differentiation-defective iPSC clones maintained high LTR expression levels, which indicated that these clones failed to complete reprogramming. lincRNA-RoR, a long intergenic non-coding RNA (lincRNA) that was previously shown to support the induction and maintenance of pluripotency, was detected among the LTR-driven transcripts. Short hairpin RNAs against the conserved sequence in LTRs or lincRNA-RoR markedly reduced the efficiency of iPSC generation. Reprogramming factors including OCT3/4, SOX2, and KLF4 bound to most LTRs. The expression of KLF4 was low in normal iPSC clones, but remained high in differentiation-defective clones. The forced expression of KLF4 in human embryonic stem cells led to the activation of LTRs and defects in neural differentiation. These results demonstrated that the transient overexpression of KLF4/LTR/lincRNA-RoR played crucial roles in reprogramming toward pluripotency in humans, whereas a failure in its re-silence resulted in differentiation defects.

overall_design: OCT3/4, SOX2 and KLF4 bindings in human dermal fibroblast and iPSC

first: Mari

last: Ohnuki

first: Kazutoshi

last: Takahashi


title: Critical role of transient activation of human endogenous retroviruses during reprogramming toward pluripotency (RNA-Seq)

overall_design: Nine samples were prepared as intermediate state of cells between human dermal fibroblast and iPSC. One iPSC clone and 4 subclones derived from defective iPSC exhibit normal differentiation ability.


title: Unbiased Capture Hi-C reveals the potential targets of breast cancer susceptibility loci

city: London

first: Nicola

last: Dryden

address: 237 Fulham Road

department: Breast Cancer Research

country: United Kingdom

institute: Institute of Cancer Research

summary: Genome-wide association studies have identified over 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions; several map to gene deserts, regions of several hundred kb lacking protein-coding genes. We hypothesized that gene deserts harbour long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21 and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long non-coding (lnc)RNAs, over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2 and MYC; target lncRNAs included DIRC3, PVT1 and CCDC26. For two gene deserts we were able to define a set of SNPs that were correlated with the published risk variant and that clustered within the bait end of an interaction peak. Preliminary functional analyses implicate one SNP (rs12613955; 2q35) as a potentially functional variant.

overall_design: Capture Hi-C was carried out in BT483, SUM44, and GM06990 cell lines to investigate breast cancer risk loci 2q35, 8q24.21 and 9q31.2.

first: Olivia

last: Fletcher

first: Laura

last: Broome

first: Sarah

middle: L

last: Maguire


title: Transient ER binding and p300 redistribution support a physiological squelching model for immediate ER-repressed genes

city: Charlottesville

first: Michael

middle: Joseph

last: Guertin

state: VA

address: 1340 Jefferson Park Ave

department: Biochemistry and Molecular Genetics

institute: University of Virginia

summary: Selective transcriptional activation and repression of genes throughout signaling cascades and development are poorly understood. Transcription factors (TF) orchestrate patterns and magnitude of transcriptional response, but TF action, or inaction, is highly dependent upon TF kinetics, distance from genes, chromatin architecture, and the local occupancy of other TFs. We integrated genomic transcription, chromosome looping, TF binding, and chromatin structure data to analyze the molecular cascade that results from estradiol-induced (E2) signaling in human MCF-7 breast cancer cells and addressed the context-specific nature of gene regulation. We analyzed kinetic ChIP-seq that profiled the master regulator of the E2-mediated response, estrogen receptor (ER), and found that transient ER binding sites are specifically associated with enhancers of repressed genes.

overall_design: We performed replicate ChIP-seq experiments prior to estrogen treatment and 2min, 5min, 10min, 40min, and 160min after E2 treatment.


title: The photoreceptor chaperone FHY1 has an independent role in gene modulation and plant development under far-red light [ChIP-seq]

city: Shenzhen

first: Bosheng

last: Li

state: Guangdong

address: SUStech Huiyuan #1 406

department: MCDB

country: China

institute: SUSTC (current, may different with previous institution )

overall_design: To determine whether phyA and FHY1 associate with the same genes upon FR irradiation, four-day old etiolated 35S: GFP-FHY1 fhy1-1 transgenic seedlings were exposed to 3hr FR. The treated plants were utilized for ChIP-seq analysis with an anti-FHY1 antibody that exhibits high specificity in the detection of FR-induced FHY1-association events. FHY1 associated-DNA samples from three distinct biological replicates and an input DNA sample (as a negative control) were subjected to high-throughput Solexa (Illumina) sequencing.

first: Fang

last: Chen

first: Jordan

last: Demone

first: Jean-Benoit

last: Charron

first: Xiarong

last: Shi

first: Xing

last: Deng


title: Required Enhancer: Matrin-3 Structure Interactions for Homeodomain Transcription Programs

status: Public on Aug 03 2014

city: San Diego

first: Qi

last: Ma

address: 9500 Gilman Dr,

institute: UCSD

summary: Study of the POU-homeodomain transcription factor, has revealed that, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer / coding gene transcriptional program.

overall_design: All ChIP-seq(s) were designed to understand the unique features, associated molecular mechanisms and functions of Matrin3 in the Homeodomain transcription study. Gro-seq Samples are used to detect gene expression between knockdown control and knockdown two key factors in our study.

first: Dorota

last: Skowronska-Krawczyk


title: Genome-wide maps of NME2 in A549 lung adenocarcinoma cells.

city: Delhi

first: Vinod

middle: Kumar

last: Yadav

state: Delhi

address: Mall Road

department: Bioinformatics

country: India

institute: IGIB

summary: It is widely believed that reorganization of nucleosomes result in availability of transcription factor (TF) binding sites for eukaryotic gene regulation. Recent findings also show TFs induced during physiological perturbations can alter nucleosome occupancy to facilitate DNA binding. Although, these suggest a close relationship between TF binding and nucleosomes, the nature of this interaction, or to what extent it influences transcription is not clear. Moreover, since physiological perturbations induced multiple TFs, relatively direct effect of any TF on nucleosome occupancy remains poorly addressed. With these in mind, we used a single TF to induce physiological changes and following characterization of the two states (before and after induction of the TF) we determined: (a) genome wide binding sites of the TF, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. We find only ~20% of TF binding results from nucleosome repositioning - interestingly, almost all corresponding genes were transcriptionally altered. Whereas, when TF-occupancy was independent of nucleosome repositioning only a small fraction of corresponding genes were expressed/repressed. These observations suggest a model where TF occupancy leads to transcriptional change only when coupled with nucleosome repositioning in close proximity. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-sequencing) typically overlaps with only a small fraction of genes that change expression.

overall_design: The nature of interaction between TF binding and nucleosomes and what extent it influences transcription

middle: K

first: Ram

last: Thakur

first: Ankita

last: Singh

first: Aradhita

last: Baral

first: Akinchan

last: Kumar

first: Shantanu

last: Chowdhury


title: ChIP-seq of RNA polymerase II, transcription factors and histone modification in retinal pigment epithelial cells

city: Cambridge

first: Richard

last: Young

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: Retinal pigment epithelial cells are critical for eye function and loss of cell function is linked to age-related blindness.  Relatively little is known about the transcriptional regulatory networks in these cells.  The datasets presented here are ChIP-seq experiments for RNA polymerase II , transcription factors and histone modifications in human retinal pigment epithelial cells.

overall_design: ChIP-Seq for transcription factors, RNA polymerase, histone modifications and CTCF in retinal pigment epithelial cells

first: Ana

middle: D

last: Alessio


title: ChIP-seq of enhancer-associated transcription factors and histone modifications

summary: The datasets presented here are transcription factors, transcriptional co-factors or histone modifications associated with active enhancers in a variety of mouse and human cell types.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Two functionally distinct subsets of mast cells generated through IL-2 independent CD25 activities

city: Vienna

first: Iros

last: Barozzi

address: Borschkegasse 8a

country: Austria

institute: Medical University Vienna

summary: CD25 (IL-2R) can be expressed on the surface of immune cells in the absence of other chains of the interleukin-2 receptor (IL-2R), which are indispensable for IL-2 signaling. We identified two novel mast cell subsets, characterized by the differential expression of surface CD25, and by the ability to produce different cytokines and to proliferate, both in vitro and in vivo. We provide evidence that functional differences between the two mast cell populations were dependent on CD25 itself, which directly modulated mast cell proliferation and responses in vivo. These effects were completely independent from IL-2 or the expression of the other chains of the high-affinity IL-2R, indicating an autonomous and previously unappreciated role for CD25 in regulating cell functions. Similar results were also obtained in dendritic cells, which are known to express CD25 but to be unresponsive to IL-2. Our findings indicate a general role for CD25 in contexts where IL-2 signaling is not involved, and may have important implications for all mast cell-related diseases, including mastocytosis, where CD25 is aberrantly expressed on pathogenic mast cells.

overall_design: Total mRNA of FACS-sorted CD25pos and CD25neg populations of primary bone marrow-derived mast cells (BMMCs) was extracted and subjected to by multiparallel sequencing.

first: S

last: Monticelli

first: G

last: Natoli

first: I

last: Polletti


title: A549 cells before/after NME2 induction

type: Genome binding/occupancy profiling by genome tiling array


title: A novel IRES identified in DMD results in a functional N-truncated dystrophin, providing a potential route to therapy for patients with 5’ mutations.

status: Public on Aug 02 2014

city: Salt Lake City

first: Robert

middle: B

last: Weiss

state: UT

department: Human Genetics

institute: University of Utah

summary:
Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on human skeletal muscle transcripts from a patient with Becker muscular dystrophy.
Methods: Tissue homogenates were prepared from frozen sections of a muscle biopsy obtained from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control.  Ribosome-protected fragments and total RNA were prepared from a single homogenate, so starting RNA populations for both libraries were closely matched.  Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads.   RPF-Seq libraries were built using the TruSeq Small RNA Sample Preparation Kit (Illumina) and RNA-Seq libraries were built using the TruSeq RNA Sample Preparation v2 Kit (Illumina). RPF-Seq and RNA-Seq libraries were subjected to 50 cycles of single-end sequencing on an Illumina HiSeq 2000 instrument.  Trimmed and filtered RPF- and RNA-Seq reads were mapped to RefSeq fasta sequences downloaded from the UCSC genome browser (hg19 assembly).
Results: Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to Duchenne muscular dystrophy.  However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that results in the expression of a highly functional N-truncated dystrophin.  This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosomal profiling.
Conclusions: Our results provide a molecular explanation for the rescue of 5’ truncating mutations via a heretofore undescribed mechanism of post-transcriptional regulation of dystrophin expression. The presence of a glucocorticoid-inducible IRES within a highly conserved region of the DMD sequence strongly suggests a programmed role for alternate translation initiation, and ongoing efforts to understand the relevant cell lineage-specific and/or conditional activation signals will shed light on underlying mechanisms of IRES control and elucidate potentially novel functions of dystrophin.

overall_design: Skeletal muscle ribosome-protected fragment and RNA-Seq profiles from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control were generated by deep sequencing using the Illumina HiSeq 2000.

first: Nicolas

last: Wein

first: Diane

middle: M

last: Dunn

first: Kevin

last: Flanigan


title: Genome-wide maps of HES1 binding-site in human chondrogenic cell.

city: Tokyo

first: Shurei

last: Sugita

address: 7-3-1, Hongo, Bunkyo-ku

department: Sensory & Motor System Medicine

institute: The University of Tokyo

summary: Notch signaling modulates skeletal formation and osteoarthritis (OA)  development through induction of catabolic factors. Here we examined functional roles of Hes1, the representative downstream transcription factor of Notch signaling, during these processes. Chromatin immunoprecipitation-sequencing (ChIP-seq) identified resposive  elements of Hes1 around  gene loci of Mmp13 and Adamts5, which were  catabolic enzymes of cartilage matrix.

overall_design: Examination of  HES1 binding site in human chondrogenic SW1353 cells.

first: Taku

last: Saito


title: TET1 regulates hypoxia-induced epithelial-mesenchymal transition through DNA demethylation (hMeDIP-Seq)

status: Public on Aug 01 2014

city: Taoyuang

first: Kou-Juey

last: Wu

state: Taiwan

address: No.15, Wenhua 1st Road, Gueishan Dist

department: Cancer Genome Research Center

country: Taiwan

institute: Chang Gung Memorial Hospital at Linkou

summary: In order to explore the status of DNA methylation in hypoxia response, we show that TET1, a DNA dioxygenase converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates hypoxia-responsive gene expression. Hypoxia/HIF-2α regulates the expression of TET1. Knockdown of TET1 mitigated hypoxia-induced EMT. RNA sequencing and 5hmC sequencing identified the set of TET1-regulated genes.

overall_design: Four samples (Four samples, Hypoxia (scrambled control), Hypoxia (TET1-si), Normoxia (scrambled control) and Normoxia (TET1-si), are performed by RNA-Seq and hMeDIP-Seq


title: ChIP-seq of cyclin-dependent kinases

summary: Several cyclin-dependent kinases (CDKs) are known to have roles in transcriptional regulation.  The datasets presented here are ChIP-seq experiments for different CDKs and RNA polymerase II in murine embryonic stem cells and Jurkat cells.

overall_design: ChIP-Seq of cyclin-dependent kinases in mouse embryonic stem cells and Jurkat human T cell acute lymphoblastic leukemia cell line

first: Jessica

last: Reddy


title: TET1 regulates hypoxia-induced epithelial-mesenchymal transition through DNA demethylation (RNA-Seq)

overall_design: Four samples (Four samples, Hypoxia (scrambled control), Hypoxia (TET1-si), Normoxia (scrambled control) and Normoxia (TET1-si), are performed by RNA-Seq and hMeDIP-Seq  RNA-Seq and hMeDIP-Seq


title: Lineage reprogramming of adult mouse liver cells and B-lymphocytes to neural stem-like cells using defined factors [ChIP-seq]

summary: The overexpression of transcription factors Oct4, Sox2, Klf4, and c-Myc reprograms a somatic nucleus to one that is transcriptionally and epigenetically indistinguishable from an embryonic stem (ES) cell. However, it is still unclear if transcription factors can completely convert the nucleus of a differentiated cell into that of a distantly related cell type such that it maintains complete transcriptional and epigenetic reprogramming in the absence of exogenous factor expression. To test this idea, we screened a library of doxycycline-inducible vectors encoding neural stem cell (NSC)-expressed genes and found that stable, self-maintaining NSC-like cells could be induced under defined growth conditions after transduction of transcription factors. These induced NSCs (iNSCs) were characterized in the absence of exogenous factor induction and were shown to be transcriptionally, epigenetically, and functionally similar to endogenous embryonic cortical NSCs. Importantly, iNSCs could be generated from multiple adult cell types including liver cells and B-cells with genetic rearrangements.  Our results show that self-maintaining proliferative neural cells can be induced from non-ectodermal cells by expressing specific combinations of transcription factors.

overall_design: ChIP-seq data from primary neural progenitor cells (Sox2-GFP) and induced neural progenitor cells were generated by deep sequencing using Illumina Hi-Seq 2000.

first: John

middle: P

last: Cassady

last: DAlessio

first: Rudolf

last: Jaenisch


title: Genome-wide DNA methylation analysis reveals dynamic changes in the cardiac methylome during post-natal heart development (RNA-Seq)

city: Melbourne

first: Mark

last: Ziemann

state: VIC

address: 85 Commercial Rd

department: Bioinformatics Working Group

country: Australia

institute: Burnet Institute

summary: Epigenetic modifications have emerged as central players in the coordination of gene expression networks during cardiac development. While several studies have investigated the role of histone modifications during heart development, relatively little is known about the role of DNA methylation. The purpose of the current study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the neonatal period, which is an important developmental window for cardiac maturation and cardiomyocyte cell cycle arrest. We used methyl binding domain protein sequencing (MBD-seq) and mRNA-seq to profile DNA methyation and gene expression respectively in neonatal hearts at P1 and P14 stages. Thousands of differentially methylated regions (DMRs) were identified between P1 and P14, the vast majority of which were hypermethylated. Gene ontology analysis revealed that these hypermethylated genes were associated with transcriptional regulation of important developmental signaling pathways, including Hedgehog, BMP, TGF beta, FGF and Wnt/b-catenin signaling. A significant enrichment for myogenic transcription factors and Smad2/3/4 binding sites was also noted among differentially methylated peaks at P14. This study provides novel evidence for widespread alterations in DNA methylation during post-natal heart maturation and suggests that DNA methylation plays an important role in cardiomyocyte cell cycle arrest during the neonatal period.

overall_design: mRNA-seq to profile gene expression in neonatal hearts at P1 and P14 stages (post-natal day 1 and 14 respectively) in three biological replicates.

first: Choon

last: Sim

first: K

middle: N

last: Harikrishnan

first: Antony

last: Kaspi

first: Jenny

last: Ooi

first: Lisa

last: Chang

first: Ishant

last: Khurana

first: Eric

last: Olson

first: James

middle: E

last: Hudson

first: Assam

last: El-Osta

first: Enzo

middle: R

last: Porrello


title: Genome-wide DNA methylation analysis reveals dynamic changes in the cardiac methylome during post-natal heart development

type: Methylation profiling by high throughput sequencing

overall_design: Refer to individual Series


title: Genome-wide DNA methylation analysis reveals dynamic changes in the cardiac methylome during post-natal heart development (MBD-Seq)

overall_design: We used methyl binding domain protein sequencing (MBD-seq) to profile DNA methyation in neonatal hearts at P1 and P14 stages (post-natal day 1 and 14 respectively) in three biological replicates.


title: Gene expression changes as a result of E-cadherin loss in an isogenic non-malignant MCF10A and MCF10A CDH1-/- breast cells

city: Dunedin

first: Augustine

last: Chen

address: 710 Cumberland Street

department: Biochemistry

country: New Zealand

institute: University of Otago

summary:
Background: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT).
Methods: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4bp deletion in CDH1 exon 11.
Results: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed.
Conclusions: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling.

overall_design: Examination of the impact of E-cadherin (CDH1) loss in an isogenic pair of breast cell lines.

first: Henry

last: Beetham

first: Michael

last: Black

first: Parry

last: Guilford


title: Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

city: Boston

first: Maxim

last: Gerashchenko

state: Massachusetts

address: 77 Louis Pasteur Ave, NRB 435

department: Medicine

institute: Brigham and Women's Hospital

summary: Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.

overall_design: Ranging concentrations of cycloheximide and various stress contitions were tested with Ribo-seq

first: Vadim

last: Gladyshev


title: Genome wide DNA methylation of Epstein-Barr virus infected immortalized normal oral keratinocytes

city: Shreveport

first: Rona

middle: S.

last: Scott

state: LA

address: 1501 Kings Highway

department: Microbiology and Immunology

institute: Louisiana State University Health Sciences Center-Shreveport

summary: The oral cavity is the persistent reservoir for EBV with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns that are regulated by epigenetic modifications involving DNA methylation and chromatin structure. Such regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may inadvertently result in long-lasting, oncogenic host epigenetic reprogramming. To test this hypothesis in the context of EBV infection of epithelial cells, we established a transient infection model to identify the epigenetic consequences after EBV infection of immortalized normal oral keratinocytes and subsequent viral loss.

overall_design: NOK cells were single cell cloned (designated as cl1) and EBV infected by co-culture with a recombinant Akata EBV strain carrying neomcycin resistance and GFP expression cassette in place of EBV BXLF1.  Cells were selected with 350 ug/ml G418.  As a selection control, cells were transfected a plasmid carrying a neomycin resistance cassette.  Cells were passaged 10 times with G418, followed by removal of selection pressure.  After an additional 10 passages, cells were single cell cloned and the presence of EBV was determined by various methods.  Three clones were identified as being EBV negative and said to be transiently infected.  Uninfected parental and vector transfected cells were passaged in the same manner and also single cell cloned.    The DNA methylation profiles were examined using reduced representation bisulfite sequencing.

middle: S


title: Widespread genetic epistasis among cancer genes.

city: Chicago

first: Kevin

last: White

institute: University of Chicago

summary: We performed NGS-based transcript profiling (RNA-seq) to profile transcripts that are expressed in MCF10A cells.  12,332 genes with FPKM>1 were considered as expressed in MCF10A cells.

overall_design: mRNA profiles of MCF10A cells were generated by deep sequencing using Illumina Hiseq 2000.

first: Xiaoyue

last: Wang


title: Mechanism of DNA methylation-directed histone methylation by KRYPTONITE

city: Seattle

first: Christopher

middle: Joel

last: Hale

state: WA

address: 1959 NE Pacific St., HSC H-458

department: Pathology

institute: University of Washington

summary: In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the epsilon-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation.

overall_design: Plants homozygous for null mutations in the KRYPTONITE H3K9 methyltransferase were stably transformed with transgenes encoding the wildtype KYP protein or transgenes carrying induced point mutations in the KYP active site. The resulting lines were assayed for DNA methylation by whole-genome bisulfite sequencing to learn the efficiency with which wildtype and mutant versions of the KYP protein could restore DNA methylation lost in a kyp mutant. Samples 7 and 8 were run as single Illumina lanes and as such were compared to a previous Col sample (GSM881756), this Col sample was realigned to the TAIR10 genome for this study and as such updated processed files are available with this submission. These samples were used to define kyp mutant CHG context DMRs that were complemented upon introduction of the wildtype KYP protein. Samples 1-6 were run as multiplexed samples and were used to assay the degree of complementation for various point mutants. All plants are in the Col ecotype background.


title: RNA-SEQ analysis of single cells extracted from the metanephric mesenchyme of E11.5 Crym-EGFP transgenic mice

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

institute: IGMM MRC Human Genetics Unit

summary: We used micro-dissection with FACS sorting techniques to isolate single cells from the metanephric mesenchyme of the Embryonic day 11.5 (E11.5) developing kidney. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database.

overall_design: Kidneys are harvested from Tg(Crym-EGFP)GF82Gsat mice.  Single cells are extracted from E11.5 metanephric mesenchyme using manual micro-dissection techniques.  A subset of these cells is analyzed individually via Fluidigm single cell analysis.  The long term goal is to generate a transcriptional atlas of the developing kidney.

first: Andrew

last: Potter

middle: W

last: Brunskill

first: Steven


title: RNA-SEQ analysis of single cells extracted from E12.5 embryonic kidneys.

summary: We used micro-dissection and trypsinization techniques to isolate single cells from the Embryonic day 12.5 (E12.5) total kidney. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database.

overall_design: E12.5 kidneys are dissected; the kidneys are made into a single cell suspension via trypsinization.  A subset of these cells is analysed individually via Fluidigm C1 single cell analysis.  The long term goal is to generate a single cell resolution transcriptional atlas of the developing kidney.

first: Eric


title: RNA-SEQ analysis of single cells extracted from the renal vesicles of P4 Crym-EGFP transgenic mice

summary: We used micro-dissection with FACS sorting techniques to isolate renal vesicle single cell types from post natal day (P4) kidneys. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database.

overall_design: Kidneys are harvested from Tg(Crym-EGFP)GF82Gsat mice.  Single cells are extracted from P4 renal vesicles using micro-dissection with FACS sorting techniques.  A subset of these cells is analyzed individually via Fluidigm single cell analysis.  The long term goal is to generate a transcriptional atlas of the developing kidney.


title: Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs [methylation]

city: Ya'an

first: Mingzhou

last: Li

state: Sichuan

address: No.46  Xinkang Road

department: College of Animal Science and Technology

institute: Sichuan Agricultural University

summary:
Background: Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.
Results: We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.
Conclusions: This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.

overall_design: We collected the longissimus dorsi muscles tissue from Jinhua pigs which aged 0.5 year and seven years and study the genome-wide DNA methylation difference between the two age periods.


title: Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation

city: South San Francisco

first: Matt

last: Grimmer

state: CA

address: 455 E Grand Ave.

department: Oncology Bioinformatics

institute: Genentech

summary: Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consist- ing of multiple zinc finger domains are currently be- ing developed for clinical applications. However, no genome-wide investigations into their binding speci- ficity have been performed. We have created six- finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB do- main (SKD) that interacts with a complex contain- ing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5- fold, with a majority of the new sites located out- side of promoters. De novo motif analyses suggest that the lack of binding specificity is due to sub- sets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger bind- ing sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.

overall_design: ATF plasmids were designed and stably integrated into MCF7 cells as described in (Stolzenburg et al. 2012).  Stable lines were grown at 30–80% confluency in Dulbecco’s Modified Eagle’s Medium (Corning, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin; cells were selected using 5 µg/ml puromycin (VWR, Radnor, PA) and 200 µg/ml G418 (VWR, Radnor, PA). ATF expression was induced by treatment with media containing 1µg/ml doxycycline (VWR, Radnor, PA) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h. ATF expression was confirmed by hemagglutinin (HA) tag western blot prior to HA ChIP-seq, histone ChIP-seq and RNA-seq analysis.  For HA-tag ChIP-seq, stable MCF7 cell lines were induced using 100 ng/ml doxycycline (Sigma) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 mL low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 × 10∧7 cells/mL. Cells are then sonicated to a fragment size range of 500–800 bp. Samples were then diluted in 1 mL low-salt buffer and incubated with 3 µL of anti-HA antibody (Covance, Princeton, NJ). Three-hundred microliter Sepharose A beads (GE Healthcare Life Science) were used for pull-down. Samples were sequenced at the UNC-CH Genome Analysis Facil- ity (Chapel Hill, NC) on an HiSeq (Illumina, San Diego, CA) to read counts of 4.1–67.3 M total reads. For histone ChIP-seq, antibodies to H3K4me3 (Cell Signaling Tech- nologies CST9751), H3K9Ac (Cell Signaling Technologies CST9649) and H3K9me3 (Diagenode pAb-056-050) were used; samples were prepared as previously described (O’Geen et al. 2011).

first: Matthew

first: Sabine

last: Stolzenburg

first: Ethan

last: Ford

first: Ryan

last: Lister

first: Pilar

last: Blancafort

first: Peggy

middle: J

last: Farnham


title: Transcriptional dysregulation of synaptic genes in a human iPSC model of major mental disorders

city: Santa Barbara

first: Kenneth

last: Kosik

address: 552 University Rd

department: Neuroscience Research Institute

institute: University of California Santa Barbara

summary: Schizophrenia and other psychiatric disorders are postulated to be developmental disorders resulting from synapse dysfunction. How susceptibility genes for major mental disorders could lead to synaptic deficits in humans is not well-understood. Here we generated induced pluripotent stem cells (iPSCs) from four members of a family in which a frame-shift mutation of Disrupted-in-schizophrenia-1 (DISC1) co-segregated with psychiatric disorders and further produced different isogenic iPSC lines via genetic editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPSC-derived forebrain neurons. Mechanistically, mutant DISC1 dysregulates the expression of many genes related to synapses and psychiatric disorders and depletes wild-type DISC1 and the NCoR1 transcription co-repressor complex. Furthermore, mechanism-guided pharmacological inhibition of phosphodiesterases rescues synaptic defects in mutant neurons. Our study uncovers a novel gain-of-function mechanism through which the psychiatric disorder-relevant mutation affects synaptic functions via transcriptional dysregulation and provides insight into the molecular and synaptic etiopathology of psychiatric disorders.

overall_design: Two patient derived iPSC lines carrying heterozygous 4bp deletion in DISC1 gene and 1 related control were analyzed in biological triplicate

first: Zhexing

last: Wen

first: HaNam

last: Nguyen

first: Ziyuan

last: Guo

last: Lalli

first: Xinyuan

first: Yijing

last: Su

first: Nam-Shik

last: Kim

first: Ki-Jun

last: Yoon

first: Jaehoon

last: Shin

first: Ce

last: Zhang

first: Georgia

last: Makri

first: David

last: Nauen

first: Elmer

last: Guzman

first: Humei

last: Yu

first: Cheng-Hsuan

last: Chiang

first: Nadine

last: Yoritomo

first: Kozo

last: Kaibuchi

last: Ross

first: Jizhong

last: Zou

first: Kimberly

last: Christian

first: Linzhao

last: Cheng

first: Russell

last: Magolis

first: Gong

last: Chen

first: Guo-li

last: Ming

first: Hongjun

last: Song


title: Profiling and comparison of miRNA populations in male and female tissues from Drosophila melanogaster at various stages

city: Cold Spring Harbor

first: Delphine

last: Fagegaltier

state: NY

address: One Bungtown Rd

department: Genomics Division

institute: Cold Spring Harbor Lab

summary: By surveying miRNA populations in each sex, we identified sets of miRNAs differentially expressed in male and female tissues across various stages of development.

overall_design: Small RNAs cloning of dissected male and female tissues from Drosophila melanogaster at various stages

first: Gregory

last: Hannon

first: Thomas

last: Gingeras


title: MicroRNA Directly Enhances Mitochondrial Translation during Muscle Differentiation.

city: Wuhan

first: Bo

last: Yang

state: Hubei

address: Luo-jia-shan, Wuchang

department: Biochemistry and Molecular Biology

institute: College of Life Sciences, Wuhan University

summary: MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by Ago2 CrossLinking ImmunoPrecipitation coupled with sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1 mediated translational stimulation in the mitochondria and repression in the cytoplasm.

overall_design: Examination of miRNA's regulation function in mitochondria in C2C12 myoblasts cells and myotubes cells with CLIP-seq (Ago2).

first: Xiaorong

last: Zhang

first: Xinxin

last: Zuo

first: Zongran

last: Li

first: Yuanchao

last: Xue

first: Yu

last: Zhou

first: Jie

last: Huang

first: Xiaolu

last: Zhao

first: Yun

last: Yan

first: Huiqiong

first: Peipei

last: Guo

first: Hui

last: Sun

first: Lin

first: Yi

first: Xiang-Dong

last: Fu


title: TET1 regulates hypoxia-induced epithelial-mesenchymal transition through DNA demethylation

first: Kou-Juey

last: Wu

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


title: NSD3-NUT Fusion Oncoprotein in NUT Midline Carcinoma identified in 1221 patient-derived cell line by RNA-seq

first: Christopher

middle: A

last: French

address: 77 Avenue Louis Pasteur 630G

institute: Brigham and Women's Hospita

summary: Discarded live tumor tissue from a metastatic focus in the patient’s lung was collected under institutional review board approval through the NUT midline carcinoma registry (www.NMCRegistry.org). From this tissue the first known NUT-variant cell line, 1221, was established. To determine the putative partner gene to NUT, we performed comprehensive RNA-sequencing on RNA purified from 1221. We identified an in-frame transcript fusing the 5’ coding sequence of NSD3 (exons 1-7) to exons 2-7 of NUT. Expression of the NSD3-NUT fusion oncoprotein was verified by immunobloting with an antibody to NUT, revealing an approximately 200kDa band that is similar in size to BRD3-NUT, but smaller than BRD4-NUT

overall_design: Identification of a NUT fusion partner using RNA extracted from live cultured 1221 cell line derived from a lung metastasis from the index case of a 13 year old female with NUT-positive NMC.

first: Erica

last: Walsh

first: Madeleine

last: Lemieux


title: The Scc2NIPBL/Scc4MAU2 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions [ChIP-seq]

city: London

first: Lidia

last: Lopez-Serra

address: 44 Lincoln's Inn Fields

department: Chromosome segregation

institute: Canser Research UK

summary: The Scc2/Scc4 complex binds to broad nucleosome-free regions in the promoters of highly expressed genes. The cohesin loader is recruited to these sites by the RSC chromatin remodeling complex

overall_design: ChIp- Sequencing analysis of the distribution of Scc2 and Scc4 on budding yeast chromosomes.An input sample was analysed as a control.

first: Gavin

last: Kelly

first: Harshil

last: Patel

first: Aengus

last: Stewart

first: Frank

last: Uhlmann


title: The Scc2NIPBL/Scc4MAU2 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions.

platform_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae

overall_design: Refer to individual Series


title: Genome wide DNA methylation and expression profiling of Epstein-Barr virus infected immortalized normal oral keratinocytes

first: Rona

last: Scott


title: The composition and function of chaperone networks in Solanum lycopersicum explored by transcriptome sequencing and microarray meta-analysis

city: Greifswald

first: Stefan

last: Simm

address: Walter-Rathenau-Straße 48

country: Germany

institute: University Medicine Greifswald

summary: Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by heat stress transcription factors (Hsfs). The role of Hsfs and Hsps in HS-response in tomato was initially examined by transcriptome analysis using the Massive Analysis of cDNA Ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points toward two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato.

overall_design: 2 samples

first: Sotirios

last: Fragostefanakis

first: Puneet

last: Paul

first: Klaus-Dieter

last: Scharf

first: Enrico

last: Schleiff


title: The Scc2NIPBL/Scc4MAU2 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions [Mnase-seq]

overall_design: MNase digestion of purified DNA followed by paired-end sequencing was performed in order to investigate genome-wide nucleosomal positioning in S. cerevisiae under multiple experimental conditions.


title: Dehydration stress memory genes of Zea mays; comparison with Arabidopsis thaliana

city: Lincoln

first: Jean-Jack

middle: M.

last: Riethoven

state: NE

address: 1901 Vine Street

department: Beadle Center for Biotechnology

institute: University of Nebraska-Lincoln

summary: Arabidopsis thaliana plants that have experienced an initial exposure to dehydration stress (“trained plants”) have an increased ability to maintain leaf relative water content (RWC) during subsequent stresses than plants experiencing the stress for the first time and transcription of  selected dehydration response genes is altered during successive exposures to dehydration stress. This physiological and transcriptional behavior of trained plants is consistent with a “memory “of an earlier stress. It is unknown whether such memory is present in other Angiosperm lineages and whether it is an evolutionarily conserved response to stress. Here, we analyzed the behavior and transcriptomes of maize (Zea mays) plants experiencing multiple dehydration stresses and compare them with responses of the evolutionarily distant A. thaliana. We found structurally related genes in maize that displayed the same memory-type  responses as in A. thaliana, providing evidence of the conservation of function and transcriptional memory in the evolution of plants’ dehydration stress response systems. Similar to A. thaliana, trained Z. mays plants retained higher RWC during dehydration stress than untrained plants, due in part to maintaining reduced stomatal conductance, despite full recovery of RWC, after the first stress. Divergent transcriptional memory responses were also expressed, suggesting diversification of function among stress memory genes. Some dehydration stress memory genes were also shared with other stress and hormone responding pathways, indicating complex and dynamic interactions between different plant signaling networks.   The results provide new insight into how plants respond to multiple dehydration stresses and provide a platform for studies of the functions of memory genes in adaptive responses to water deficit in monocot and eudicot plants .

overall_design: For each condition (water, S1, and S3) the transcriptome was sequenced for two replicates. The watered condition is considered the control.

first: Laetitia

last: Virlouvet

first: Yong

last: Ding

first: Ning

last: Liu

first: Sabina

last: Russo

first: Michael

last: Fromm

first: Zoya

last: Avramova


title: PR-SET7 inactivation causes hepatocyte necrosis and spontaneous development of hepatocellular carcinoma derived from ductal progenitor cells

city: Vassilika Vouton, Herakleion

first: Iannis

last: Talianidis

state: Crete

address: 100 N. Plastira Str

department: Institute of Molecular Biology and Biotechnology

country: Greece

institute: Foundation for Research and Technology - Hellas

summary: PR-SET7-mediated histone-4 lysine-20 methylation has been implicated in mitotic condensation, DNA damage response and replication licencing. Here we show that PR-SET7 function in the liver is pivotal for maintaining genome integrity. Hepatocyte-specific deletion of PR-SET7 in mouse embryos resulted in G2 arrest followed by massive cell death and defect in liver organogenesis. Inactivation at postnatal stages caused cell duplication-dependent hepatocyte necrosis with unusual features of autophagy, termed "endonucleosis". Necrotic death was accompanied by inflammation, fibrosis and compensatory growth induction of neighboring hepatocytes and resident ductal progenitor cells. Prolonged necrotic-regenerative cycles coupled with oncogenic STAT3 activation replaced pre-existing hepatocytes with hepatocellular carcinoma derived entirely from ductal progenitor cells. Hepatocellular carcinoma in these mice displays a cancer stem cell gene signature specified by the co-expression of ductal progenitor markers and oncofetal genes.

overall_design: Mice carrying hepatocyte specific inactivation of PR-SET7 were generated in order to investigate the function of PR-SET7 histone methyl transferase in liver organogenesis, hepatocyte proliferation and liver regeneration. P15 WT mice were injected intra-peritoneally (ip) with 25ml per kg DEN (diethyl nitrosamine). Mice were examined for RNA expression at 8 months old.

first: Kostas

last: Nikolaou

first: Panagiotis

last: Moulos

first: George

last: Chalepakis

first: Pantelis

last: Hatzis

first: Hisanobu

last: Oda

first: Danny

last: Reinberg


title: Identification of miRNAs regulated by TGF-b in human CD8+ T cells

city: Würzburg

first: Anoop

last: Chandran P

address: Josef-schneiderstrasse

institute: University of Würzburg

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus


title: Identification of miRNAs regulated by TGF-b in human CD8+ T cells (small RNA-seq)

summary: The goal of the current study was to identify miRNAs regulated by TGF-b in human CD8+ T cells and analyze the function of these miRNAs in shaping the immunomodulatory effect of TGF-b in these cells. We identified the miR-23a cluster to be upregulated and found that this cluster could target key molecules (IFN-g and LAMP1) involved in immune response by CD8+ T cells.

overall_design: CD8+ T cells were isolated and purified from healthy human peripheral blood of 5 donors and were activated using beads coupled to anti-CD2, anti-CD3 and anti-CD24. They were then treated with 5ng/ml TGF-b, with 1µM SD-208 or left untreated. RNA from these cells were then isolated and used for deep sequencing.

last: Chandran

first: Andreas

last: Keller

first: Jörg

last: Wischhusen

first: Norbert

last: Eichner

first: Gunter

last: Meister


title: Normal gene expression variation in Enchytraeus albidus - transcriptional responses to control conditions

city: Aveiro

first: Sara

last: Novais

address: Campus de Santiago, Department of Biology, University of Aveiro

department: Biology

country: Portugal

institute: CESAM & University of Aveiro

summary: Several questions remain on how ecologically relevant can a change in gene expression be or how to quantify after which changes are beyond normal biological variation. Knowing genes are highly sensitive and self-regulating pieces can we design our experiments to minimize natural variability sources? E.g., are results from experiments performed at different times always comparable? Further, the effect of carriers (solvents) is often neglected. The main aim of the present paper was to create critical discussion on these and other questions, while providing suggestions for improvement and interpretation. We have analysed gene data sets comparing standard control test conditions: 1) from similar experiments performed at different occasions in time; 2) to culture media, 3) to acetone control, 4) from various exposure periods (2, 4 and 8 days) and 5) between replicates. In general, the transference of organisms from culture media to standard testing suggests a lowered general metabolism. Responses to acetone seem to be related with changes in soil structure, usually associated with hypoxia conditions. Coefficient of variance (CV) between biological replicates was >0.1 for 17% of the genes being as high as 1.3. These results represent the first contribution for the establishment of a normal operating range (NOR) for E. albidus gene expression. Further, comparison between various control exposure times showed significant variation - organisms have a “time-like” transcription signature. Tests may be designed to potentially reduce these variability sources, although, this will partly be inherent to the species, and most important is to understand the ground for interpretations.

overall_design: Gene expression in E.albidus was measured in several control samples. The experimental design consisted in three analyses to infer: 1) the effect of transferring animals from culture conditions onto standard control test conditions: control (CT) versus culture (CULT); 2) the effect of acetone (frequently used as carrier) compared to water: acetone (ACT) versus water control (CT); 3) the effect of different exposure time in CT conditions: CT4d and CT8d vesus CT2d

middle: C

first: Mónica

last: Amorim


title: Identification of miRNAs in developing seeds of Brassica napus

city: Porto Alegre

first: Rogerio

last: Margis

state: RS

address: Av. Bento Goncalves 9500

department: Centro de Biotecnologia

country: Brazil

institute: Universidade Federal do Rio Grande do Sul

first: Ana

last: Körbes

first: Frank

middle: F

last: Guzman

first: Rogério


title: E2a is necessary for Smad2/3 dependent transcription and the direct repression of lefty

city: Stanford

first: Andrea

middle: E.

last: Wills

address: 300 Pasteur Drive

department: Genetics

institute: Stanford School of Medicine

summary: We characterized the binding of Smad2/3 using ChIP-SEQ in both control gastrula-stage X. tropicalis embryos and embryos depleted of the transcription factor E2a (E2a-MO).  We also characterized gene expression in control and E2a-depleted embryos by RNA-Seq.  For ChIP-Seq, three biological replicates of E2a-MO were performed, and two biological replicates of Control embryos were performed.  For RNA-Seq, two biological replicates of E2a-MO were performed, and two biological replicates of Control embryos were performed, and the mean expression levels of each gene were compared.

overall_design: For ChIP-Seq, three biological replicates were performed for E2a-depleted X. tropicalis embryos, and two biological replicates were performed for control gastrula-stage embryos.  For RNA-Seq, two biological replicates were performed for both E2a-depleted embryos and control embryos, and the mean expression levels were compared for each gene.

first: Julie

last: Baker


title: Hypoxia-related Transcription factor ChIP-Seq data in T47D breast cancer cells

status: Public on Jul 31 2014

city: Chapel Hill

first: Qing

last: Zhang

address: 450 West Drive

institute: University of North Carolina at Chapel Hill

summary: We report the comprehensive genome-wide binding peaks for key factors inovled in oxygen sensing pathways, such as HIF1α, HIF1β and EglN2. In addition, we also report the genome-wide binding peaks for NRF1 in breast cancer cells

overall_design: We conducted HA-EglN2, HIF1α, HIF1β (ARNT) or NRF1 ChIP-Seq in the T47D cell line that overexpresses HA-EglN2 in the presence of hypoxia (1%) and DMOG treatment. T47D parental cells  treated with the same condition followed by  HA ChIP-seq served as the control to filter non-specific binding.

first: Chengyang

last: Wang


title: Hypoxia-related transcription factor ChIP-seq and EglN2 knockdown expression profiling in T47D breast cancer cells


title: In depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms

city: Durham

first: Adam

middle: Wesley

last: Whisnant

state: NC

address: 213 Research Drive 427 CARL Bldg

department: Molecular Genetics and Microbiology

institute: Duke University

summary: The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4+ PBMCs and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced crosslinking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by pre-existing cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites.

overall_design: Small RNA profiling of HIV-1 infected cells through total small RNA deep sequencing and identification of RNA-induced silencing complex (RISC)-associated HIV-1 sequences through the RNP-immunoprecipitation technique of PAR-CLIP on RISC

first: Hal

last: Bogerd

first: Omar

last: Flores

first: Phong

last: Ho

first: Jason

middle: G

last: Powers

first: Natalia

last: Sharova

first: Mario

last: Stevenson

first: Chin-Ho

last: Chen

first: Bryan

last: Cullen


title: Targeting Unique Metabolic Properties of Breast Tumor Initiating Cells

first: Andrew

last: Gentles

address: 1201 Welch Road

institute: Stanford University

summary: Bulk cancer cell populations are known to display distinct metabolic properties compared to their normal counterparts. However, relatively little is known about heterogeneity of metabolic properties within tumors. In this study we show that, analogous to some normal stem cells, breast tumor initiating cells (TICs, also called cancer stem cells) have distinct metabolic properties compared to non-tumorigenic cancer cells (NTCs). Transcriptome profiling using RNA-Seq revealed TICs under-express genes involved in mitochondrial oxidative phosphorylation and although TICs are relatively quiescent, they preferentially perform glycolysis over oxidative phosphorylation compared to NTCs. TICs contain fewer mitochondria and display lower expression and activity of pyruvate dehydrogenase (Pdh), a key regulator of oxidative phosphorylation. Metabolic reprogramming of TICs by pharmacologic activation of Pdh preferentially eliminates TICs in vitro and in vivo. Our findings reveal unique metabolic properties of TICs and indicate that metabolic reprogramming represents a promising strategy for targeting these cells.

overall_design: Examination transcriptome profiles for breast tumor initiating cells (TICs) and non-tumorigenic cells (NTCs)

first: Weiguo

last: Feng

first: Ramesh

middle: V

last: Nair

first: Max

last: Diehn


title: Expression of orthologs of ribosome biogenesis factors in vegetative and reproductive tissues in Solanum lycopersicum

first: Stefan

last: Simm

department: Bioinformatics

summary: Ribosome biogenesis is a major cellular processes involving a large inventory of proteinaceous and RNA cofactors. Recent bioinformatics approaches discovered a large but not complete overlap of the inventory between yeast, mammals and plants. We re-inspected the complexity of ribosome biogenesis factors (RBFs) by assigning co-orthologs to each protein family in the A. thaliana and S. lycopersicum genomes. We realized that about 25% of all analyzed RBFs in the two plant genomes are encoded by more than one gene. The majority is expressed in leaves and anthers of tomato as determined by next generation sequencing (NGS). For the analysis of NGS data we developed a software pipeline ‘CRACPipe’. This pipeline is designed to be very flexible with respect to genome selection and experimental set up. The NGS analysis revealed the expression of 24.538 genes in tomato leaves and of 22.893 genes in anthers. The observed expression of RBFs was confirmed by qRT-PCR for a randomly chosen sub-set of genes. Further, analysis of the expression profile of selected RBFs during male gametophyte development revealed a differential expression in the different developmental stages for the majority of genes. Based on our analysis we conclude that RBFs described in yeast but not present in plant genomes as concluded from previous orthology analysis are in parts replaced by other RBFs for which multiple genes have been identified in here. In addition, the obtained complexity for some RBFs accounts for a tissue and developmental specific regulation of ribosome biogenesis as well.

first: Sotirios

last: Fragostefanakis

first: Puneet

last: Paul

last: Keller

first: Jens

last: Einloft

first: Klaus-Dieter

last: Scharf

first: Enrico

last: Schleiff


title: Identifying conserved and novel miRNAs in developing seeds of Brassica napus using deep sequencing [mRNA-seq]

first: Rogerio

last: Margis

summary: MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. A total of 62 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally, 32 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. The contigs from the assembled mRNA-seq data allowed for a search for putative new precursors and led to the identification of 13 novel miRNA families. Differential expression between the libraries was determined through a statistical analysis of normalized miRNA reads and revealed several miRNAs and isomiRNAs that were more abundant during the developing stages. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comprehensive study of the miRNA transcriptome of B. napus seeds and will provide a basis for future research on more targeted studies of individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus.

overall_design: RNA profiles in 2 different seed libraries (mature seeds and a pool of developing seed stages) of Brassica napus by deep sequencing (Illumina HiSeq2000).

first: Ana

last: Körbes

first: Frank

last: Guzman

first: Rogério


title: Nucleosome occupancy changes in mammalian cell differentiation and reprogramming (Mnase-Seq)

city: Boston

first: Robert

last: Kingston

address: 185 Cambridge Street

institute: Massachusetts General Hospital

summary: Embryonic stem cells (ESCs) and induced-pluripotent stem cells (iPSCs) self-renew and differentiate into an array of cell types in vitro and in vivo. A complex network of genetic and epigenetic pathways regulates the self-renewal and differentiation of these pluripotent cells, and the structure and covalent modifications of chromatin play a prominent role in this process. We examine nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach that enabled the identification of regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. The majority of changes in nucleosomal signatures that occur in differentiation are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of pluripotency and likely identify key regulatory regions that play a role in determining cell identity.

overall_design: Micrococcal nuclease digestion of chromatin in crosslinked cells was followed by high throughput sequencing. These experiments were carried out in four mouse cell types: embryonic stem cells, induced pluripotent stem cells, somatic tail-tip fibroblasts and liver, and three human cell types: H1-OGN embryonic stem cells, H1-OGN induced pluripotent stem cells, and fibroblasts differentiated from H1-OGN ESCs. At least two replicates performed with each cell type were sequenced.

first: Jason

last: West

first: April

last: Cook

first: Burak

middle: H

last: Alver

first: Mattias

last: Stadtfeld

first: Aimee

middle: M

last: Deaton

first: Konrad

last: Hochedlinger

first: Peter

last: Park

first: Michael

middle: Y

last: Tolstorukov


title: The developmental potential of iPSCs is greatly influenced by the selection of the reprogramming factors

status: Public on Jul 30 2014

city: La Jolla

first: Joseph

middle: R

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4 and Myc (OSKM) into somatic cells. Though iPSCs are pluripotent, they frequently exhibit high variation in their quality as measured by chimera contribution and tetraploid (4n) complementation. Thus, improving the quality of iPSCs is an indispensable prerequisite for future iPSC-based therapy. Here we show that one major determinant for iPSCs quality is the combination of reprograming factor selected. Ectopic expression of Sall4, Nanog, Esrrb and Lin28 (SNEL) in MEFs efficiently generated high quality iPSCs as compared to other combinations of factors. SNEL-iPSCs produced approximately 5 times more efficiently “all-iPSC” mice compared to OSKM-iPSCs. While differentially methylated regions, transcript number of master regulators, establishment of ESC-specific super enhancers, and global aneuploidy were comparable between the lines,  aberrant expression of 1,765 genes, trisomy of chromosome 8 and abnormal H2A.X deposition were frequently observed  in poor quality OSK-iPSCs and OSKM-iPSCs.

overall_design:
Whole-genome single-base resolution MethylC sequencing collected from a variety of Mouse pluripotent cells
To reveal the DNA methylation signature associated with developmental competence, we selected the following groups of iPSC lines for whole-genome bisulfite sequencing:
i) "Poor quality" iPSCs: This group included the three OSKM-iPSC lines Nanog-GFP OSKM#2, Oct4-GFP OSKM#2 and KH2 OSKM(Stadtfeld et al., 2010), that either did not produce fully developed pups or produced very low number of pups;
ii) "Good quality" iPSCs: This group included BC_2 OSKM (Carey et al., 2011) and Nanog-GFP SNEL#3, both of which gave rise to live, normal pups that survived only few hours;
iii) "High quality" iPSCs consisting of Nanog-GFP SNEL#2 and Oct4-GFP SNEL#1, both of which generated live pups that survived postnatally (representative pups from each iPSC group are shown in Figure S3A).
iv) As controls we used Nanog-GFP, Oct4-GFP and KH2 (Beard et al., 2006) ESCs. Note that all cells were cultured in 2i medium but not in mES medium.

first: Yupeng

last: He

first: Matthew

middle: D

last: Schultz

last: Nery


title: Nucleosome occupancy changes in mammalian cell differentiation and reprogramming

middle: E


title: RNAseq analysis of NGATHA loss and gain of function alleles

city: Milan

first: Vicente

last: Balanza

state: Lombardia

address: Via Celoria 26

department: Bioscienze

country: Italy

institute: University of Milan

summary: The four NGATHA genes (NGA) form a small subfamily within the large family of B3-domain  transcription factors  of  Arabidopsis thaliana.  NGA  genes  act redundantly to direct  the development of  the apical tissues of the  gynoecium, the style and the stigma.  Previous studies indicate that  NGA  genes  could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium  through the upregulation  of  two  different  YUCCA  genes, which encode flavin monooxygenases involved in auxin biosynthesis. We  have compared three developing pistil transcriptome data sets from wildtype, nga  quadruple mutants and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA  genes  as major  regulators of auxin accumulation and distribution within  the developing gynoecium.

overall_design: Expression profile comparation of wild type, nga mutant and NGA overexpression

last: Balanzà

first: Cristina

last: Ferrandiz

first: Lucia

last: Colombo


city: Cambridge

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

overall_design: ChIP-Seq for Med1 in reprogrammed and partially reprogrammed mouse embryonic stem cell lines and corresponding controls

first: Yosef

last: Buganim

first: Styliana

last: Markoulaki

first: Niek

last: van Wiemartschen

first: Heather

last: Hoke

first: Tao

last: Wu

first: Kibibi

last: Ganz

first: Batool

last: Akhtar-Zaidi

first: Brian

last: Abraham

first: David

last: Porubsky

first: Elisabeth

last: Kulenkampff

first: Dina

last: Faddah

first: Linyu

last: Shi

first: Qing

last: Gao

first: Sovan

last: Sarkar

first: Malkiel

last: Cohen

first: Johanna

last: Goldmann

first: Andrew

last: Xiao

last: Lansdorp

first: Rudolf

last: Jaenisch


title: Genome-wide targets of conserved histone chaperone Asf1 in budding yeast

city: Hyderabad

first: Pooran

middle: Singh

last: Dewari

state: Andhra Pradesh

address: Uppal Road

department: Transcription Lab

institute: Centre for Cellular and Molecular Biology

summary: Asf1, through its histone chaperone activity, helps chromatin closing/opening during DNA replication, repair, recombination and transcription. Despite extensive research on Asf1-mediated physiological functions, a genome-wide localization map is lacking, limiting our knowledge of chromosomal features targeted by Asf1. We present a high-resolution genome-wide map of Asf1, localizing at essentially all pol III-transcribed genes, highly active pol II-transcribed genes and heterochromatic features. Pol III-transcribed genes are negatively regulated by Asf1, whereas pol II genes are regulated indirectly by Asf1-dependent H3K56 acetylation. Interestingly, Asf1 localization along yeast chromosomes shows nearly identical distribution to that of the condensin complex, predicting a functional overlap in chromosome architecture and genome organization.

overall_design: ChIP-seq analysis of Asf1 targets using a yeast strain that expresses an 18-Myc tag fused to the C-terminus of ASF1. Two biological replicates and one mock/control were performed. The Illumina GAII was used. ChIP-seq reads are aligned to the budding yeast sacCer3 (2011) assembly.

middle: S

first: Purnima

last: Bhargava


title: Gene expression profiling associated with knockdown of LKB1 in human intrahepatic cholangiocarcinoma

status: Public on Jul 29 2014

city: Duarte

first: keqiang

last: zhang

address: 1500 East Duarte Road

department: Molecular Pharmacology

institute: City of Hope National Medical Center

summary: The experiment was designed to display differential gene expression profiling in three human intrahepatic cholangiocarcinoma (ICC) cells upon knockdow of LKB1 tumor suppressor, by using RNAseq technology.

overall_design: LKB1 was first attenuated in three ICC cells: HuH-28, RBE, and SSP-25 by siRNA-mediated knockdown. Total RNA was extracted from duplicated cancers cells transfected with control siRNA and LKB1 specific siRNA at 48h posttransfection for RNAseq analysis. Differentially expressed genes in LKB1-attenuated ICC cells were identified in comparison to that in ICC cells transfected with control siRNA.

first: K

last: Zhang

first: Y

last: Yen

first: J

last: Wang

last: Lu

first: X

last: Wu


title: High resolution ChIP sequencing reveals novel bindings targets and prognostic role for SOX11 in Mantle cell lymphoma

status: Public on Aug 07 2014

city: New York

first: Samir

last: Parekh

address: 1470 Madison Ave, 5th FL, Room 114

department: Hematology and Medical Oncology

institute: Icahn School of Medicine at Mount Sinai

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

overall_design: Refer to individual Series


title: RNA sequencing (RNA-SEQ) of Dnmt1 and Dnmt3a conditional knockout mice

first: Seungyeul

last: Yoo

address: 1425 Madison Avenue

department: Genetics and Genomic Sciences

summary:
Purpose: Epigenetic regulation contributes to pathogenesis of neurondegenerative disease.  We found that dis-regulation of a  242-gene subnetwork related to DNA methylation regulated neuronal differentiation are common in AD and HD. There are two DNA methyltransferases, DNMT1 and DNMT3A, in the subnetwork. DNMT1 is one of top hub genes in the network and likely to play a key regulatory role in the subnetwork. To validate that DNMT1 is a key regulator for the subnetwork, and DNMT3A not as one, we constructed two brain-specific conditional knockout mice.
Methods: Cortices were dissected from Dnmt1 conditional knockout (CKO), Dnmt3a CKO, and respective littermate control mice at 18 weeks. RNA was isolated from three biological replicates for each genotype using TRIzol (Invitrogen) extraction and isopropanol precipitation. RNA samples were resuspended in water and further purified with RNeasy columns with on-column DNase treatment (Qiagen). RNA purity was assessed by measuring the A260/A280 ratio using a NanoDrop and RNA quality checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument according to the manufacturer's instructions. Sequence reads were aligned to mouse genome assembly mm10 and quantified using Cufflinks.
Results: Genes differentially expressed in CKO mice compared to littermate control mice, or the Dnmt1 CKO signature, including GSN (the genes with the largest number of connection in the subnetwork) and SOX10 (a key transcription factor of myelination), significantly overlaps with the subnetwork. Broadly, the Dnmt1 CKO signature is enriched for genes involved in GO biological processes immune response, lipid metabolism, endocytosis, glial cell differentiation, and nerve ensheatment, consistent with biological function of the subnetwork. Moreover, the Dnmt1 CKO mice show increased predilection to seizures, an incidence also increased in patients with AD (Amatniek et al, 2006) as well as juvenile form of HD (Cloud et al, 2012). In contrast, the Dnmt3a CKO signature does not overlap with the subnetwork (p=0.07) and is not enriched for any GO biological process.
Conclusions: These results suggest the Dnmt1 regulates common genes related with AD and HD while Dnmt3a has little effect.

overall_design: All of the mice used in this study were handled in accordance with IACUC-approved protocols. Dnmt1flox/flox (Fan et al, 2001; Jackson-Grusby et al, 2001) and Dnmt3aflox/flox (Nguyen et al, 2007) mice were backcrossed onto a C57BL/6 background and crossed with Olig1-cre mice to generate Dnmt1 conditional knockout (Olig1cre/+;Dnmt1flox/flox) and littermate control (Olig1+/+;Dnmt1flox/flox) mice, and Dnmt3a conditional knockout (Olig1cre/+;Dnmt3aflox/flox) and littermate control (Olig1+/+;Dnmt3aflox/flox) mice.

first: Zhu

last: Jun

first: Casaccia

last: Patrizia


title: MicroRNA transcriptome in weaning and sucking porcine intestines

city: Hangzhou

first: Xin

last: Tao

address: Shiqiao Road 145

institute: Zhejiang Academy of Agricultural Sciences

summary: MicroRNAs (miRNAs) play important roles in intestinal diseases; however, the role of miRNAs during weaning stress is unknown. In our study, six jejunal small RNA libraries constructed from weaning piglets at 1, 4 and 7 d after weaning (libraries W1, W4 and W7, respectively) and from suckling piglets on the same days as the weaning piglets (libraries S1, S4 and S7, respectively) were sequenced using Solexa high-throughput sequencing technology. Overall, 260 known swine miRNAs and 317 novel candidate miRNA precursors were detected in the six libraries. The results revealed that 16 differentially expressed miRNAs were found between W1 and S1; 98 differentially expressed miRNAs were found between W4 and S4 (ssc-mir-146b had the largest difference and ssc-mir-215 had the highest expression level); and 22 differentially expressed miRNAs were found between W7 and S7. Sequencing miRNA results were validated using RT-qPCR. Approximately 12,819 miRNA-mRNA interactions corresponding to 4,250 target genes were predicted. The biological analyses revealed that the differentially expressed miRNAs regulated small intestinal metabolism, stressful responses, cellular and immune functions and miRNA biosynthesis in piglets. Therefore, the small intestine miRNA transcriptome was significantly different between weaning and suckling piglets; the difference varied with the number of days after weaning.

overall_design: six small RNA libraries from weaning piglets at 1, 4 and 7 d after weaning and from suckling piglets on the same days as the weaning piglets, respectively.  For every small RNA library construction, 4 biological total RNA samples isolated from each treatment and control were separately pooled with equal contribution.

first: Ziwei

last: Xu


title: Identification of tRNA sequences in Lactococcus lactis

city: Guangzhou

first: Gong

address: Huang-Pu Avenue West 601

department: Institute of Life and Health Engineering

institute: Jinan University

summary: we report the identification and sequences of the tRNAome of industrially relevant microorganism Lactococcus lactis

sample_organism: Lactococcus cremoris subsp. cremoris MG1363

overall_design: Three Next Generation sequencing runs annotated as S1, S2 and S3 were performed. Cells were harvested at exponential phase and tRNA was isolated. S1 and S2 were spiked with Phe-tRNAGAA from yeast and Lys-tRNAUUU from E. coli prior to cell lysis. S3 was spiked with Phe-tRNAGAA from yeast and Lys-tRNAUUU from E. coli before the library preparation to estimate the possible loss of tRNA in the extraction process.

first: Pranav

last: Puri

first: Bert

last: Poolman

first: Pieter

last: van der Vlies


title: Su(Hw) and Shep RIP-seq in Kc and BG3 cell lines

city: Bethesda

first: Ryan

last: Dale

state: MD

address: Rm 10D39, 10 Center Drive

department: National Institute of Child Health and Human Development

institute: National Institutes of Health

overall_design: RIP-seq with multiple antibodies to two proteins in two Drosophila cell lines, at least 2 replicates for each IP and input

middle: K

first: Elissa

middle: P

last: Lei


title: Small RNA profile in Moso Bamboo root and leaf

city: Oxford

first: Irina

last: Mohorianu

address: Headington

department: Medical Sciences Division

institute: University of Oxford

summary: The small RNA libraries from Moso bamboo (Phyllostachy heterocycla) roots and leaves were constructed by using high definition adapters .  The small RNA profiles were analyzed. A collection of micro RNAs with similarity to the micro RNA entries in mirbase were discovered. The putative genomic loci of the micro RNAs were identified.

overall_design: Analysis of small RNA profiles from the root and leaf tissues of young Moso Bamboo seedlings

first: Ping

first: Li

last: Yang

first: Hansheng

last: Zhao

first: Zhimin

last: Gao

first: Tamas

last: Dalmay


title: Potential Influence of Staphylococcus aureus Clonal Complex 30 Genotype and Transcriptome on Hematogenous Infections

city: Rochester

first: Steven

last: Gill

address: Box 672, Elmwood Avenue

department: Microbiology and Immunology

institute: University of Rochester School of Medicine and Dentistry

summary:
We report the application of RNA-Seq to assess genomic transcriptional profiles of S. aureus clinical isolates from Clonal Complex 30 and related genetic backgrounds. In this study, CC30 isolates were shown to be significantly less virulent than other CCs in two in vivo sepsis models. The association of CC30 with adhesin-based
complications in humans  but not sepsis-induced mortality in animal models may be due in part to its unique genomic transcriptional profile and suggests that specific genotypes of S. aureus may be more likely to elicit certain complication types.

overall_design: Staphylococcus aureus transcriptome profiles of exponential growth phase in vitro cultures were generated by RNA-Seq in duplicate, using Illumina HiSeq2500.


title: A new dataset of spermatogenic vs oogenic transcriptomes in the nematode C. elegans

city: Madison

first: Marco

middle: Antonio

last: Ortiz

state: Wisconsin

address: 433 Babcock Drive

department: Biochemistry

institute: University of Wisconsin-Madison

summary: The nematode Caenorhabditis elegans is an important model for studies of germ cell biology, including specification as sperm or oocyte, the meiotic cell cycle and gamete differentiation.  Fundamental to those studies is a genome-level knowledge of the germline transcriptome.  Here we use RNA-Seq to identify genes expressed in isolated XX gonads, which are roughly 95% germline and 5% somatic gonadal tissue.  We generate data from mutants making either sperm [fem-3(q96)] or oocytes (fog-2), both grown at 22°C.  Our dataset identifies a total of 10,754 mRNAs in the polyadenylated transcriptome of XX gonads, with 1,723 enriched in spermatogenic gonads, 2,869 enriched in oogenic gonads and the remaining 6,274 not enriched in either.  These spermatogenic, oogenic and gender-neutral gene datasets compare well with those of earlier studies, but double the number of genes identified.  We also query our RNA-Seq data for differential exon usage and find 351 mRNAs with sex-specific isoforms.  We suggest that this new dataset will prove useful for studies focusing on C. elegans germ cell biology.

overall_design: Comparison of spermatogenic vs oogenic transcriptomes

middle: A

first: Daniel

last: Noble

first: Elena

last: Sorokin

first: Judith

last: Kimble


title: Novel Bioinformatics Method for Identification of Genome-Wide Non-Canonical Spliced Regions using RNA-Seq Data

city: Ann Arbor

first: Maureen

last: Sartor

state: MI

address: 100 Washtenaw Avenue

institute: University of Michigan

summary: We developed a bioinformatics approach called the Read-Split-Walk (RSW) pipeline, and evaluated it using two Ire1α heterozygous and two Ire1α-null samples. The 26nt non-canonical splice site in Xbp1 was detected as the top hit by our RSW pipeline in heterozygous samples but not in the negative control Ire1α knockout samples. We compared the Xbp1 results from our approach with results using the alignment program BWA, STAR, Exonerate and the Unix “grep” command.  We then applied our RSW pipeline to RNA-Seq data from the SKBR3 human breast cancer cell line. RSW reported a large number of non-canonical spliced regions for 108 genes in chromosome 17, which were identified by an independent study.

overall_design: Identification of non-canonical spliced regions for mouse MEF samples (two Ire1α heterozygous and two Ire1α-null samples)

first: Yongsheng

last: Bai

first: Justin

last: Hassler

first: Randal

middle: J

last: Kaufman


title: Functionally diverse dendritic mRNAs rapidly associate with ribosomes following a novel experience

first: Leon

last: Reijmers

state: MASSACHUSETTS

address: 136 Harrison Ave

department: Neuroscience

institute: Tufts University

summary: The subcellular localization and translation of messenger RNA (mRNA) supports functional differentiation between cellular compartments. In neuronal dendrites, local translation of mRNA provides a rapid and specific mechanism for synaptic plasticity and memory formation, and might be involved in the pathophysiology of certain brain disorders. Despite the importance of dendritic mRNA translation, little is known about which mRNAs can be translated in dendrites in vivo and when their translation occurs. Here we collect ribosome-bound mRNA from the dendrites of CA1 pyramidal neurons in the adult mouse hippocampus. We find that dendritic mRNA rapidly associates with ribosomes following a novel experience consisting of a contextual fear conditioning trial. High throughput RNA sequencing followed by machine learning classification reveals an unexpected breadth of ribosome-bound dendritic mRNAs, including mRNAs expected to be entirely somatic. Our findings are in agreement with a mechanism of synaptic plasticity that engages the acute local translation of functionally diverse dendritic mRNAs.

overall_design: RNA-Seq of ribosome-bound mRNA immunoprecipitated from dendrites and soma of CA1 pyramidal neurons in the mouse hippocampus

first: Joshua

last: Ainsley


title: Genetic disruption of Ctcf destabilizes DNA methylation

first: Matt

last: Teater

address: 445 E 69th St

institute: Weill Cornell Medical College

summary: Ctcf heterozygous knockout mice are susceptible to neoplasia in a broad range of tissues, including lymphoma, endometrial cancer, and non-small cell lung cancer.  Retention of the wild type Ctcf allele in these tumors establishes CTCF as a haploinsufficient tumor suppressor gene.  Both human tumors and normal murine tissues with CTCF disruption are characterized by genome-wide differences in DNA methylation relative to CTCF wild type tissues, indicating even modest disruption of CTCF broadly destabilizes DNA methylation in vivo.  This cross species functional analysis identifies CTCF as a commonly mutated tumor suppressor gene and establishes a central role for DNA methylation stability in tumor suppression.

overall_design: RRBS sequencing of transgenic Ctcf heterozygous mice and wild-type litter mate whole lung tissue.

first: Christopher

last: Kemp

first: Ari

last: Melnick

first: Galina

middle: N

last: Filippova


title: MicroRNA expression profiling of the fifth-instar posterior silk gland of Bombyx mori

city: HangZhou

first: Boxiong

last: Zhong

state: Zhejiang province

address: Yuhangtang Road 866

institute: Zhejiang University

summary:
Background: The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production.
Results: Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, from the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the genes into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing.
Conclusion: The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs in silk production.

overall_design: Sequencing 10 total RNA samples from the posterior silk gland of different strains and developmental stage using Illumina Solexa technology.  Four strains of silkworm (Q, B, QB and BQ) with different two development stages (stage 1: fourth instar molting to day 2 of fifth instar; stage 2: fifth instar day 3 to day 8 before spinning, according to our previous genes expression cluster analysis), and two strains (R1 and J1) from entire period (stage 1 + stage 2).

first: Jisheng

last: Li

first: Yimei

last: Cai

first: Lupeng

last: Ye

first: Shaohua

last: Wang

first: Jiaqian

last: Che

first: Zhengying

last: You

first: Jun

last: Yu