title: Complexity of Murine Cardiomyocyte miRNA Biogenesis, Sequence Variant Expression and Function

status: Public on Jul 21 2012

city: Acton

first: Hardip

middle: Rameshbhai

last: Patel

state: Australian Capital Territory

address: Bld 131, ANU Campus,

department: Genome Biology Department

country: Australia

institute: The Australian National University

summary: microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ~40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5′ start but common at the 3′ end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5′ isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5′ isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5′ most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.

overall_design: We examined miRNA profiles of mouse HL1 cells in culture at 4 time points (3 in biological duplicate)

first: David

last: Humphreys

first: Carly

last: Hynes

middle: R

first: Grace

middle: H

last: Wei

first: Leah

last: Cannon

first: Diane

last: Fatkin

first: Catherine

middle: M

last: Suter

first: Jennifer

middle: L

last: Clancy

first: Thomas

last: Preiss


title: Chromatin Interactions by 5C from ENCODE/University of Washington

status: Public on Jul 19 2012

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

country: USA

institute: ENCODE DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:sull@u.washington.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

This track contains chromatin interaction data from the University of Washington ENCODE group generated using 5C (Chromatin Conformation Capture Carbon Copy). The 5C method is used here to define short and long-range range interactions between transcription start sites (TSS) and DNaseI hypersensitive sites (DHS) or other genomic features. The 5C method is summarized below.
Transcription factors bind to promoter-associated proteins, bringing the associated DNA sequences in close proximity to each other. Cross linking the DNA and proteins immobilizes these interactions and thus maintains their close proximity. Cleavage of the sample with restriction endonuclease followed by ligation results in hybrid molecules where a fragment with a regulatory element is physically associated with a fragment containing a TSS. The interactions are then detected by oligonucleotide-dependent, ligation-mediated assays, where one set of primers is complementary to the end of fragments with a TSS and the second set of primers are complementary to fragments with a feature. Primers are designed to the forward strand of the feature and the reverse strand of the TSS so that ligation only occurs between TSS and feature, not between different features. Specific interactions are detected by massively parallel sequencing.
The data in this track comprises two different experiment types focusing on targeted regions:
Gene-targeted project: Analysis of DNase I hypersensitive sites reveals many genes where there are multiple sites restricted to the cell type where a protein is observed to be expressed. These sites potentially identify regulatory sites for the gene. This set of experiments attempts to observe interactions between these DHS sites and transcription starts in 25 regions selected based on genes expressed in GM06990 (B-lymphocyte), BJ (foreskin fibroblast), HepG2 (liver cancer cell line), or SK-N-SH_RA (neuroblastoma cell line, SKNSH, differentiated with retinoic acid).
Myc project: Genome wide association studies have identified SNPs linked to prostate, colon, and breast cancer in the gene desert region upstream of the myc gene. 5C of HindIII fragments interacting with those containing refSeq txStarts in this region were performed in 5 cell types: GM12878 (B-lymphocyte), CaCo2 (colon cancer cell line), LNCaP (prostate cancer cell line), MCF7 (breast cancer cell line), and K562 (erythroleukemia cell line).

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
Cells were grown according to the approved ENCODE cell culture protocols. The isolated nuclei were formaldehyde cross-linked. The DNA isolated from the nuclei was cleaved with restriction enzyme, ligated, and cross-links removed to create a 3C library (Dekker et al., 2002). Primers complementary to the TSS and feature were added, annealed and ligated to produce a 5C library (Dostie et al., 2006). The DNA fragments generated in the ligation mediated-reactions were partially digested with DNaseI, end-repaired and ligated to adapters, before sequencing. The sequencing reads generated were mapped to the predicted ligation products. The number of sequences mapping to predicted junction fragments were tabulated from sequencing runs. The number of times a sequence was detected for a given interaction between a TSS and feature indicates the relative strength of the interation.
Gene-targeted project: Forward primers were designed to HindIII sites in a 230-415 kb sequence centered on the DNase I hypersensitive sites of interest. Reverse primers were designed to HindIII sites for all transcription starts extending 1 Mb on either side of the region targeted by the forward primer set. Matrix files are labeled by the coordinates of the region covered by the forward primer set. These experiments were done in a multiplex manner with the forward and reverse primers for all 25 regions mixed together in a single reaction. Two replicates were performed for 4 cell lines for 25 regions. High-throughput sequencing was performed on an ABI SOLiD instrument collecting 50 bp reads. The interaction files provided map all the reads in the output sequence without a mismatch threshold.
Myc project: Forward primers were designed to HindIII fragments of 4.29 Mb section of human chromosome 8 centered on the gene desert 5~R of the myc gene. Reverse primers were designed to all HindIII fragments containing refseq txStarts in a 7.6 Mb region extending > 2 Mb on either side of the forward primer set. High-throughput sequencing was performed on an ABI SOLiD instrument collecting 50 bp reads. The interaction files provided map all the reads in the output sequence without a mismatch threshold.
Data were verified by sequencing biological replicates displaying correlation coefficient > 0.9.

first: Richard

last: Sandstrom


title: Transcript structures in Shewanella oneidensis MR-1

city: Berkeley

first: Morgan

middle: N

last: Price

address: 1 Cyclotron Road Mailstop 977-152

department: Physical Biosciences Division

institute: Lawrence Berkeley Lab

type: Expression profiling by high throughput sequencing

summary: 5' RNA-Seq of mRNA from S. oneidensis MR-1 grown aerobically in defined lactate medium

overall_design: One lane of sequence for a 5' RNASeq library from RNA treated with exonuclease to remove degraded transcripts, with single-end 40-nt reads

first: Wenjun

last: Shao

first: Adam

last: Deutschbauer

middle: P

last: Arkin


title: Chromatin Interaction Analysis Paired-End Tags (ChIA-PET) from ENCODE/GIS-Ruan

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Yijun Ruan mailto:ruanyj@gis.a-star.edu.sg). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

This track was produced as part of the ENCODE Project. It shows the locations of protein factor mediated chromatin interactions determined by Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) data (Fullwood et al., 2010) extracted from five different human cancer cell lines (K562 (chronic myeloid leukemia), HCT116 (colorectal cancer), HeLa-S3 (cervical cancer), MCF-7 (breast cancer), and NB4 (promyelocytic)). A chromatin interaction is defined as the association of two regions of the genome that are far apart in terms of genomic distance, but are spatially proximate to each other in the 3-dimensional cellular nucleus.
Additionally, ChIA-PET experiments generate transcription factor binding sites. A binding site is defined as a region of the genome that is highly enriched by specific Chromatin ImmunoPrecipitation (ChIP) against a transcription factor, which indicates that the transcription factor binds specifically to this region. The protein factors displayed in the track include estrogen receptor alpha (ERa), RNA polymerase II (RNAPII), and CCCTC binding factor (CTCF).

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) is a global de novo high-throughput method for characterizing the 3-dimensional structure of chromatin in the nucleus. In the ChIA-PET protocol, samples were cross-linked and fragmented, then subjected to chromatin immunoprecipitation. The DNA fragments that were brought together by the chromatin interactions were then proximity-ligated. During this proximity-ligation step, the half-linkers (created by the fragmentation) containing flanking MmeI sites (type IIS restriction enzymes) were first ligated to the DNA fragments and then ligated to each other to form full linkers. Full linkers bridge either two ends of a self-circularized fragment, or two ends of two different chromatin fragments. The material was then reverse cross-linked, purified and digested with MmeI. MmeI cuts 20 base pairs away from its recognition site. Tag-linker-tag (paired-end tag, PET) constructs were sequenced by ultra-high-throughput methods (Illumina or SOLiD paired-end sequencing). ChIA-PET reads were processed with the ChIA-PET Tool (Li et al., 2010) by the following steps: linker filtering, short reads mapping, PET classification, binding site identification, and interaction cluster identification. The high-confidence binding sites and chromatin interaction clusters were reported.
Chromatin interactions identified by ChIA-PET have been validated by 3C, ChIP-3C, 4C and DNA-FISH (Fullwood et al., 2009).

first: Guoliang

last: Li

first: Xiaoan

last: Ruan

first: Kuljeet-Singh

last: Sandhu

first: Fabianus

last: Mulawadi

first: Huay-Mei

last: Poh

first: Yufen

last: Goh

first: Su-Qin

last: Peh

first: Wing-Kin

last: Sung

first: Yijun

first: Raymond

last: Auerbach

first: Michael

last: Snyder


title: Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells [RNA-Seq]

status: Public on Jul 16 2012

city: Boston

first: Peter

middle: J

last: Park

state: MA

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary: Variation in chromatin composition and organization often reflects differences in genome function. Histone variants, for example, replace canonical histones to contribute to regulation of numerous nuclear processes including transcription, DNA repair and chromosome segregation. Here we focus on H2A.Bbd, a rapidly evolving variant found in mammals but not in invertebrates. We report that in human cells, nucleosomes bearing H2A.Bbd form unconventional chromatin structures enriched within actively transcribed genes and characterized by shorter DNA protection and nucleosome spacing. Analysis of transcriptional profiles from cells depleted for H2A.Bbd demonstrated widespread changes in gene expression with a net down-regulation of transcription and disruption of normal mRNA splicing patterns. In particular, we observed changes in exon inclusion rates and increased presence of intronic sequences in mRNA products upon H2A.Bbd depletion. Taken together, our results indicate that H2A.Bbd is involved in formation of a specific chromatin structure that facilitates both transcription and initial mRNA processing.

overall_design: RNA-seq was used to examine changes in gene expression upon shRNA-assisted depletion of H2A.Bbd and H2A.Z histone variants in HeLa cells. The cells treated with shRNA with no homology to the human genome were used as control.

middle: Y

last: Tolstorukov

first: Joseph

middle: A

last: Goldman

first: Cristele

last: Gilbert

first: Vasily

last: Ogryzko

first: Robert

middle: E

last: Kingston


title: Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells [ChIP-Seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Nuclei were digested with Mnase and histone-DNA complexes were isolated with antibody.


title: Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells

summary:
Variation in chromatin composition and organization often reflects differences in genome function. Histone variants, for example, replace canonical histones to contribute to regulation of numerous nuclear processes including transcription, DNA repair and chromosome segregation. Here we focus on H2A.Bbd, a rapidly evolving variant found in mammals but not in invertebrates. We report that in human cells, nucleosomes bearing H2A.Bbd form unconventional chromatin structures enriched within actively transcribed genes and characterized by shorter DNA protection and nucleosome spacing. Analysis of transcriptional profiles from cells depleted for H2A.Bbd demonstrated widespread changes in gene expression with a net down-regulation of transcription and disruption of normal mRNA splicing patterns. In particular, we observed changes in exon inclusion rates and increased presence of intronic sequences in mRNA products upon H2A.Bbd depletion. Taken together, our results indicate that H2A.Bbd is involved in formation of a specific chromatin structure that facilitates both transcription and initial mRNA processing.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Chromosome positioning and the clustering of functionally related loci in yeast is driven by chromosomal interactions

city: Auckland

first: Justin

middle: M.

last: O'Sullivan

address: 85 Park Road, Grafton

department: Liggins Institute

country: New Zealand

institute: University of Auckland

summary: In recent years there has been considerable and growing interest in the 3-dimensional organization of genomes. In yeast this has included detailed FISH studies that show a surprising degree of spatial clustering of the tRNA genes, the localization of telomeres, centromere positioning, and global studies of chromosomal interactions. In this study we performed an integrated biophysical – molecular study to produce the first high-resolution 3-dimensional reconstruction of a eukaryotic genome from first principles. Our conformations were produced using a using a molecular dynamics simulator to model the 16 yeast chromosomes as individual polymers. Critically, the compaction, folding and spatial organization of these chromosomes was informed by empirical data determined using proximity-based ligation. Our models allow us to investigate the structural organization and functional spatial clustering of the yeast nucleus at a resolution that has not previously been possible. We clearly show that yeast chromosomes have preferred yet non-exclusive positions. Surprisingly, while there are general trends relating chromosome position along the spindle pole body – nucleolus axis and chromosome size, some chromosomes have territories that contain two linked domains. Similarly, there is clear evidence for functional clustering of tRNAs and Gal4 protein binding sites. Intriguingly, these functional clusters are dynamic and the identity of the component elements alters in different model solutions.

overall_design: Genome Conformation Capture has been performed on exponentially growing Saccharomyces cerevisiae in media containing Glucose

first: Lutz

last: Gehlen

first: Gerd

last: Gruenert

first: M

middle: B

last: Jones

first: Chris

middle: D

last: Rodley

first: Jörg

last: Langowski

first: J


title: Genome-wide Detection of Genes Targeted by Aberrant Somatic Hypermutation in Lymphoma

city: New York

first: Yanwen

last: Jiang

state: NY

institute: Weill Cornell Medical College

summary: We report a novel approach to identify genome-wide somatic hypermutation hotspots from short Illumina H3K4me3 ChIPseq reads in diffuse large B-cell lymphoma cells (DLBCL).  Abberant somatic hypermation are known to occur at the promoters of several proto-oncogenes in DLBCL.  To identify such events genome-wide, we performed H3K4me3 ChIPseq experiments (as to enrich promoter sequences of actively transcribed genes) in 2 DLBCL cells lines (OCI-Ly1 and OCI-Ly8) and their normal B-cell counterparts, Naive B cells (NBC) and Germinal Center B cells (GCBs).  We discover new genes that harbor mutations in their promoter regions that are potentially introduced by the aberrant activation-induced cytosine deaminase activity in lymphoma cell lines, and many of these genes are important for the B cell biology.  Moreover, we show that these mutations can affect the activities of these promoters.  Our study provides a feasible approach for the detection of promoter mutations and broadens our knowledge on promoter mutations in lymphomas.

overall_design: Examination of 1 histone mark (H3K4me3) in 4 different cell types.


title: Identification of miRNA from Cyprinus carpio by high-throughput sequencing and bioinformatics analysis

city: Beijing

middle: T

last: Li

department: The Centre for Applied Aquatic Genomics

country: China

institute: The Centre for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences,

type: Non-coding RNA profiling by high throughput sequencing

summary: Common carp is one of the main commercial fishes captured and cultured worldwide. Although common carp genome is not finished yet, this study provides a first large scale cloning and characterization of common carp miRNAs and their potential targets. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in gene regulation of common carp.

overall_design: We constructed a small RNA library from 17 Cyprinus carpio samples.

first: Y

last: Zhu


title: Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes

city: Neuherberg

first: Giovanni

last: Pilloni

address: Ingolstädter Landstr. 1

country: Germany

institute: Helmholtz-Centre Munich

summary: The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the application of amplicon pyrosequencing. The possibility of barcode-tagged sequencing of templates gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing and, as in the early days of genetic community fingerprinting, pros and cons are continuously provided. In this study we investigate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of natural microbiota. Moreover, via quantitatively defined template spiking to the natural community, we explore the potential for recovering specific template ratios within complex microbial communities. For this reason, we pyrotag sequenced three biological replicates of three samples, each belonging from yearly sampling campaigns of sediment from a tar oil contaminated aquifer in Düsseldorf, Germany. Furthermore, we subjected one DNA extract to replicate technical analyses as well as to increasing ratios (0, 0.2, 2 and 20%) of 16S rRNA genes from a pure culture (Aliivibrio fisheri) originally not present in the sample. Unexpectedly, taxa abundances were highly reproducible in our hands, with max standard deviation of ~3% abundance across biological and ~2% for technical replicates. Furthermore, our workflow was also capable of recovering A. fisheri amendmend ratios in reliable amounts (0, 0.29, 3.9 and 23.8%). These results highlight that pyrotag sequencing, if done and evaluated with due caution, has the potential to robustly recapture taxa template abundances within environmental microbial communities.

sample_organism:
Aliivibrio fischeri
environmental samples
environmental samples
environmental samples
aquifer metagenome

platform_organism:
Aliivibrio fischeri
environmental samples
aquifer metagenome

overall_design: 9 Biological and 3 technical replicates were evaluated, as well as potential to recover qPCR-defined ratios of DNA, in 454 pyrotag sequencing

middle: S

last: Granitsiotis

first: Marion

last: Engel

first: Tillmann

last: Lueders


title: Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

status: Public on Jul 12 2012

city: Palo Alto

first: shuo

last: gu

address: 269 Campus Drive

department: Pediatrics

institute: Stanford University

summary: Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics.

overall_design: Various Argonautes associated small RNA profiles were generated by deep sequencing the Agos-IP samples in HEK293 Cells transfected with corresponding Argonaute.

first: Shuo

last: Gu

first: Mark

last: Kay


title: Genome-wide binding sites of Hypoxia inducible factor 1 (HIF1) and histone modifications

city: 4-6-1, Komaba, Meguroku

first: Takahide

last: Kohro

state: Tokyo

country: Japan

institute: LSBM, RCAST

summary: We report the high-throughput profilings of HIF1 and histone modifications in human umbilical vein endothelial cells (HUVEC). By obtaining over two billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of HUVEC under normoxia and hypoxia. We find that HIF1binds to not only to transcriptional starting sites but also enhancer regions and that HIF1 binding sites were overlapped with lysine 4 trimethylatio, monomethylation and lysine 27 acetylation . Finally, we show that chromatin state can change under hypoxia by using chromatin conformational capture assay. This study provides novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.

overall_design:
Examination of HIF1 and 3 different histone modifications in HUVEC under 2 conditions.

Related gene expression data is provided in GSE35932.

first: Imari

last: Mimura

first: Yasuharu

last: Kanki

first: Shogo

last: Yamamoto

first: Yoichiro

last: Wada

first: Tatsuhiko

last: Kodama


title: Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection.

status: Public on Jul 17 2012

city: ACTON

first: Bala

last: Thumma

state: ACT

address: Clunies Ross st

department: Plant Industry

institute: CSIRO

summary:
Purpose: Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection

Methods: We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Reads mapping to predicted transcripts were analysed with edgeR pacakge to study differntial expression of genes under control and water-stress conditions. SNPs within the genes were obtained using pileup file generated with samtools and the SNP frequencies were analysed with VarScan package. SNPs were further characterised as sysnonymous and non-synonymous using popoolation2 pacakge.

Results: Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with total gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments.

Conclusions: Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study.

overall_design: Leaf mRNA of 12 samples were used to study the gene expression under control and water-stress conditions. Three samples under water-stress conditions and three samples under control conditions were used as biological replicates for differenctial gene expression between the treatments. High-throughput sequence reads from mRNA samples were generated by deep sequencing with Illumina GAIIx.


title: Hog1 controls global reallocation of RNA Pol II upon osmotic shock

status: Public on Jul 15 2012

city: Cambridge

first: Kristen

last: Cook

address: 52 Oxford Street, NW Labs Rm 442

department: Molecular and Cellular Biology

institute: Harvard University

summary: When challenged with osmotic shock, S. cerevisiae induces hundreds of genes, despite a global reduction in transcriptional capacity.  The mechanisms that regulate this rapid reallocation of transcriptional resources are not known.  Here we show that redistribution of RNA Pol II upon stress requires the stress-responsive MAP kinase Hog1.  We find that Hog1 and RNA Pol II co-localize to open reading frames that bypass global transcriptional repression, and that these targets are specified by two osmotic stress-responsive transcription factors.  The combination of reduced global transcription with a gene-specific override mechanism allows cells to rapidly switch their transcriptional program in response to stress.

overall_design:
ChIP-sequencing of S. cervisiae RNA Pol II, Hog1, Sko1 and Hot1
Processed data file descriptions:
ORFcounts.txt: table of summed ChIP-seq reads that align to each ORF (normalized by reads per kilobase per million)
promoter_counts.txt: table of summed ChIP-seq reads that align to each promoter (1kb upstream, normalized by reads per kilobase per million)
downstream_counts.txt: table of summed ChIP-seq reads that align 3' regions (50-500 bp downstream, normalized by reads per kilobase per million)
Sko1_peak_list.txt: table of peaks found by PeakSeq
Hot1_peak_list.txt: table of peaks found by PeakSeq

first: Erin

middle: K

last: O'Shea


title: In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs

status: Public on Jul 13 2012

first: A

last: Bhutkar

address: 77 Massachusetts Avenue

institute: MIT

summary: Dicer is an RNase III-family endoribonuclease and haploinsufficient tumor suppressor that is required for the biogenesis of miRNAs, yet in vivo structure-function characterization of its RNase IIIA and IIIB domains have not been reported.  In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs.  Inactivation of the RNase IIIA or IIIB domain blocked maturation of miRNAs derived from the 3’ or 5’ arms of miRNA precursors, respectively, and resulted in altered miRNA expression profiles.

overall_design: Small RNAs from murine mesenchymal stem cells (MScs)  with and without Dicer (WT:Dicer f/f, KO:Dicer -/-, KO transfected with various hsDicer point mutants) were analyzed.

first: Allan

last: Gurtan

first: Victoria

last: Lu

first: Arjun

first: Phillip

last: Sharp


title: Cytoplasmic Assembly and Selective Nuclear Import of Arabidopsis ARGONAUTE4/siRNA Complexes

first: Yijun

last: Qi

address: #7 Science Park Road, Zhongguancun Life Science Park

institute: National Institute of Biological Sciences

summary: Here we profiled small RNAs from whole cell,  cytoplasmic and nuclear extracts from three-week-old Arabidopsis seedlings. We unexpectedly found that nuclear functional hc-siRNAs are predominantly present in the cytoplasm.

overall_design: Samples from Arabidopsis thaliana whole cell,  cytoplasmic and nuclear extracts with 3 replicates for each.  9 samples in all.

first: Ruiqiang

last: Ye

first: Wei

last: Wang

first: Yang

last: Wu


title: Differential microRNA response to a high-cholesterol, high-fat diet in livers of low and high LDL-C baboons

status: Public on Jul 14 2012

city: Winston-Salem

first: Genesio

last: Karere

state: NC

address: 1 medical center blvd

department: Internal Medicne

institute: Wake Forest Baptist Health Sciences

summary: Dysregulation of microRNAs (miRNAs) expression has been implicated in molecular genetics events leading to the progression and development of atherosclerosis. We hypothesized that miRNA expression profiles differ between baboons with low and high serum low-density lipoprotein cholesterol (LDL-C) concentrations in response to diet, and that a subset of these miRNAs regulate genes relevant to dyslipidemia and risk of atherosclerosis.

overall_design: We generated small RNA libraries from baboons differing in their LDL-C response to dietary fat and cholesterol (low LDL-C, n = 3; high LDL-C, n = 3) using liver biopsies collected before and after a high-cholesterol, high-fat (HCHF) challenge diet.  We sequenced the libraries using Next-Generation Illumina sequencing methods, analyzed the data using mirTools software and identified 517 baboon miRNAs: 490 homologous to human and 27 novel miRNAs.  HCHF diet elicited expression of more miRNAs compared to baseline (chow) diet for both low and high LDL-C baboons. Seventeen miRNAs exhibited significant differential expression in response to HCHF diet in high LDL-C baboons compared to nine miRNAs in low LDL-C baboons. Putative miRNA targets were identified with TargetScan/Base tools.  miRNAs significantly targeted more genes in high LDL-C baboons compared to low LDL-C responders.  Further, we identified miRNA isomers and other non-coding RNAs that were differentially expressed in response to the challenge diet.Our discovery of differentially expressed baboon miRNAs and their targets is a fundamental step in understanding the role of non-coding RNAs in the modulation of dsylipidemia.

first: Jeremy

last: Glenn

first: John

last: VandeBerg

first: Laura

last: Cox


title: Cucumber transcriptome tells story of early fruit development from fruit set through exponential growth

city: East Lansing

first: Kevin

last: Carr

state: MI

address: Plant Biology Lab, 612 Wilson Rd., Rm. S-20 A

department: Research Technology Support Facility

institute: Michigan State University

summary: Early stages of fruit growth from initial set through exponential growth are critical determinants of size and yield, however, there has been little detailed analysis of this phase of development.  In this study we combined morphological analysis with 454 pyrosequencing to study gene expression changes occurring in young cucumber fruit at five ages from anthesis through the end of exponential growth.  The resulting 1.13 million ESTs were assembled into 27,859 contigs with a mean length of 834 base pairs and a mean of 67 reads per contig.  Principal component analysis separated the fruit ages into three groups corresponding with cell division/pre-exponential growth (0 and 4 days post pollination (dpp)), peak exponential expansion (8dpp), and late/post-exponential expansion stages of growth (12 and 16 dpp). Transcripts predominantly expressed at  0 and 4 dpp included homologs of histones, cyclins, and plastid and photosynthesis related genes.  The group of genes with peak expression at 8dpp included cytoskeleton, cell wall, and lipid metabolism related genes and  phloem specific proteins. This group also was dominated by genes with unknown function or without Arabidopsis homologs, suggesting unique factors contributing to the rapid growth phase in cucurbits.  A second shift in gene expression was observed at 12-16dpp, which was characterized by abiotic and biotic stress related genes and significant enrichment for stress related- and developmental- transcription factor gene homologs.  Collectively, the gene expression information coupled with morphological analyses tells a progressive story of cell division, development of photosynthetic capacity, cell expansion and fruit growth, phloem activity, protection of the fruit surface, and finally transition away from fruit growth toward defense and maturation.

overall_design: mRNA profiling of early fruit development using 454 sequencing.

first: Kaori

last: Ando

first: Rebecca

last: Grumet


title: Modulation of mucosal immune responses to Clostridium difficile by peroxisome proliferator-activated receptor γ and microRNA-146b

status: Public on Jul 11 2012

city: Blacksburg

first: Pawel

last: Michalak

state: VA

institute: Virginia Bioinformatics Institute

summary:
BACKGROUND: miRNA have been shown to play an important role during immune-mediated diseases such as inflammatory bowel disease. The aim of this study was to assess differential expression of miRNA between uninfected and infected mice with Clostridium difficile strain VPI 10463
RESULTS: MicroRNA (miRNA)-sequencing analysis indicated that miR-146b, miR-1940, and miR-1298 were significantly overexpressed in colons of C. difficile-infected mice

overall_design: Colon of uninfected and C.difficile-infected C57BL6/J WT mice were sampled at day 4 post-infection with Clostridium difficile VPI 10463. The infection dose was 107 cfu/mouse.

first: Josep

last: Bassaganya-Riera

first: Raquel

last: Hontecillas

first: Monica

last: Viladomiu


title: Small RNA analysis of Piwi-induced permanently silent transgenic strains

city: Groningen

first: Eugene

last: Berezikov

address: Antonius Deusinglaan 1

department: European Research Institute for the Biology of Ageing

country: Netherlands

institute: University Medical Center Groningen

summary: Small RNA libraries from total RNA isolated from  adult animals

overall_design: Small RNA libraries were derived from genetically identical strains carrying a 21Usensor transgene in single copy. In one strain the transgene has become permanently silent and is not reactivated by RNAi against prg-1 (RNAe). In the other the transgene reactivates upon RNAi against prg-1.

first: Rene

middle: F

last: Ketting


title: Small RNA profiling of wildtype and Eri1-deficient mouse T cells

city: San Francisco

first: Molly

middle: Fisher

last: Thomas

address: 513 Parnassus Ave

department: Microbiology and Immunology

institute: University of California San Francisco

summary: Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here we show that mice deficient in Eri1, a conserved 3’-to-5’ exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK cell development and maturation. Eri1–/– NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse cytomegalovirus (MCMV) infection. Ly49H+ NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1–/– T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1–/– T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK cell development and anti-viral immunity.

overall_design: Small RNA profiling from wildtype and Eri1-deficient mouse CD4+ T cells

first: K

last: Ansel


title: High-thoughput Illumina RNA sequencing to identify downstream target genes of RABBIT EARS (RBE) in the flowers of Arabidopsis thaliana

city: West Haven

first: Francesc

last: Lopez

state: CT

department: Department of Genetics

institute: Yale University

summary: In order to identify putative downstream target genes of RBE, we sequenced mRNA from dexamethasone (DEX) and mock treated transgenic Arabidopsis line 35S:GR-RBE (RBE coding region fused to a glucocorticoid receptor domain driven by the constitutive 35S promoter) floral tissues. We compared the results from DEX and mock treatments and focused on the 832 genes whose expression was significantly reduced (P < 0.025) by 2-fold or more in DEX as compared to mock-treated plants. In this analysis, we identified MIR164c (EEP1) as a candidate target of RBE, which was further confirmed by other molecular and genetic analyses. Regulation of MIR164c by RBE is important for normal floral organ formation in Arabidopsis.

overall_design: We used two biological replicates, each with two technical replicates for four hour DEX or mock treated floral tissues to produce 8 sequencing libraries.

first: Tengbo

last: Huang

last: López-Giráldez

first: Jeffrey

last: Townsend

first: Vivian

last: Irish


title: Transcriptome and methylome interactions in rice hybrids

status: Public on Jul 10 2012

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

type: Methylation profiling by high throughput sequencing

summary: This research reports genome-wide measurements of genetic and epigenetic patterns of inheritance through an integrative analysis of BS-seq, RNA-seq, and siRNA-seq data in two inbred parents of the Nipponbare (NPB) and Indica (93-11) variety of rice and their hybrid offspring.

sample_organism: Oryza sativa Indica Group x Oryza sativa Japonica Group

sample_organism: Oryza sativa Japonica Group x Oryza sativa Indica Group

overall_design: We generated integrative maps of whole genome cytosine methylation profiles (BS-Seq), transcriptional profiles (RNA-seq), and small RNA profiles (sRNA-seq) to characterize two rice subspecies, Oryza sativa spp japonica (Nipponbare) and Oryza sativa spp indica (93-11) and their two reciprocal hybrid offspring using Illumina's sequencing-by-synthesis (SBS) platform .

first: Chodavarapu

last: Ramakrishna

first: Suhua

last: Feng

first: Bo

last: Ding

first: Stacey

last: Simon

first: David

first: Yulin

last: Jia

first: Guo-Liang

last: Wang

middle: C

first: Steve

last: Jacobsen

first: Matteo

last: Pellegrini


title: Live cell monitoring of periodic gene expression in synchronous human cells identifies forkhead genes involved in cell cycle control

status: Public on Jul 09 2012

city: Hanover

first: Gavin

last: Grant

state: NH

address: 7400 Remsen

department: Genetics

institute: Geisel School of Medicine

summary: We report the genome wide DNA binding patterns of wild type FOXK1 in asynchronous HeLa cells using chromatin precipitation followed by high-throughput sequencing (ChIP-seq).  We find that FOXK1 is bound to the promoter of a number of cell cycle genes including the E2F binding partner TFDP1.  This study is the first reported study of FOXK1 binding on a genome-wide scale in HeLa cells.

overall_design: Chromatin precipitation

first: Michael

middle: L

last: Whitfield


title: TAL1 complex in human T-cell acute lymphoblastic leukemia (RPMI-8402)

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: The oncogenic transcription factor TAL1/SCL is aberrantly overexpressed in over 40% of cases of T-cell acute lymphoblastic leukemia (T-ALL), emphasizing the importance of the TAL1-regulated transcriptional program in the molecular pathogenesis of T-ALL. Here we identify regions occupied by TAL1 and its regulatory partners HEB, E2A, and GATA3 in a T-ALL cell line (RPMI-8402).

overall_design: Human T-ALL cells were cross-linked with formaldehyde for 20 min. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract (WCE) was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total TAL1 (Santa Cruz SC-12984), TCF12/HEB (Santa Cruz SC-357),TCF3/E2A (Santa Cruz SC-349X), and GATA3 (Santa Cruz SC-22206).

first: Lee

middle: N

last: Lawton


title: Transcriptome of the demosponge Amphimedon queenslandica at life cycle transitions

city: Santa Barbara

first: Cecilia

last: Conaco

state: California

address: BioII

department: Neuroscience Research Institute

institute: University of California at Santa Barbara

summary: Poriferans, like the demosponge Amphimedon queenslandica, are the earliest diverging metazoans and may hold the key to understanding the evolution of metazoan pathways. A. queenslandica exhibits a biphasic lifecycle, with free-swimming larvae and sessile adult stages. To study changes in the transcriptome during this period of morphological and ecological transition, we generated poly(A) fragment libraries for A. queenslandica at four developmental stages: precompetent and competent larvae, postlarvae, and adult. These libraries were sequenced using Applied Biosystems SOLiD technology. This study provides the most comprehensive analysis to date of gene expression in the sponge. It has elucidated the genes that define the two main phases of the sponge lifestyle and identified genes that are important for competence and metamorphosis. More importantly, this study has provided insights into the expression of the genes that characterize metazoan features, such as cell adhesion and differentiation, in an early metazoan.

overall_design: 4 stages sequenced: precompetent larvae, competent larvae, postlarvae, adult

first: Kenneth

last: Kosik

first: Hongjun

last: Zhou


title: Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus

city: Rio de Janeiro

first: Tatiane

middle: Franca

last: Silva

state: Rio de Janeiro

address: Professo Rodolpho Paulo Rocco

department: Virologia

country: Brazil

institute: Universidade Federal do Rio de Janeiro

summary: In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some group of viruses. However, nothing is known for members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, the population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae, is characterized.Deep sequencing of small RNAs (sRNAs) from CLRDV-infected cotton leaves was performed. Results showed 21-nt to 24-nt long vsRNAs matching all the viral genome, with a higher frequency of matches in the 3â region. Equivalent amounts of sense and antisense vsRNAs were found, and the 22-nt long small RNA class was the most prominent one. Looking for cotton Dcl transcripts levels during infection, we could observe that Dcl4 seems to be up-regulated, while Dcl2 seems to be down-regulated.This is the first report on the profile of sRNAs coming from a plant infected with a member of the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAsOur results indicate that secondary structures of the viral RNAs are not the main source of the viRNAs observed, as suggested for other viruses. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpected high accumulation of 22-nt viRNAvsRNAs are discussed. CLRDV is the causal agent of worldwide cotton pathology named Cotton blue disease. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

overall_design: Total RNA obtained from leaves of the cotton plant 5 days post-infection with Cotton leafrol dawrf virus (CLRDV) compared to not-infected control.

first: T

first: E

last: Romanel

first: R

last: Andrade

first: L

last: Farinelli

first: M

last: Osteras

first: C

last: Deluen

last: Correa

middle: G

last: Schrago

last: Vaslin


title: Independence of Repressive Histone Marks and Chromatin Compaction during Senescent Heterochromatic Layer Formation (ChIP-Seq)

first: Rafik

last: Salama

address: Li Ka Shing Centre, Robinson Way

country: United Kingdom

institute: Cancer Research UK

summary: The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into non-overlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of pre-senescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

overall_design:
ChIP-seq for different histone marks in both growing and Ras-induced senescent fibroblasts, in the presence or absence of certain sh-RNAs
K9me3Grow2.bed (growing) Chip Seq Analysis of H3K9me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT
K9me3Sen2.bed (senescent) Chip Seq Analysis of H3K9me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment
K9me2Grow3.bed (growing) Chip Seq Analysis of H3K9me2 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment
K9me2Sen3.bed (senescent) Chip Seq Analysis of H3K9me2 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT
K27me3Sen3.bed (senescent) Chip Seq Analysis of H3K27me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT
K27me3Grow2.bed (growing) Chip Seq Analysis of H3K27me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment
K36me3Grow2.bed (growing) Chip Seq Analysis of H3K36me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment
K36me3Sen2.bed (senescent) Chip Seq Analysis of H3K36me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT
K4me3Grow2.bed (growing) Chip Seq Analysis of H3K4me3 in ER:Ras expressing IMR90 human diploid fibroblasts with no treatment
K4me3Sen3.bed (senescent) Chip Seq Analysis of H3K4me3 in ER:Ras expressing IMR90 human diploid fibroblasts d6 4OHT

first: Masashi

last: Narita


title: HDAC3 requirement for the inflammatory gene expression program in macrophages

city: Vienna

first: Iros

last: Barozzi

address: Borschkegasse 8a

country: Austria

institute: Medical University Vienna

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

overall_design: Refer to individual Series

first: Xuefen

last: Chen

first: Alberto

last: Termanini

first: Elena

last: Prosperini

first: Flore

last: Mietton

first: Gianluca

last: Matteoli

first: Scott

last: Hiebert

first: Gioacchino

last: Natoli


title: Genome sequencing and transcriptional profiling of the insect biocontrol fungus Beauveria bassiana

city: Shanghai

first: Chengshu

last: Wang

address: 300 Fenglin Road

department: Institute of Plant Physiology and Ecology

institute: Shanghai Institutes for Biological Sciences, CAS

summary: Insect pathogenic fungus Beauveria bassiana in one of the best studied insect biocontrol fungus, which infects insects by cuticle penetration. After breaking the cuticles, the fungus will propagate in insect hemocoel and kill insect hosts. It has also been found that the mycelia of B. bassiana can penetrate plant tissues to reach insect inside plant, e.g. corn borer (Ostrinia furnacalis), but do not cause damage to plants. The mechanism of fungal physiological plasticity is poorly understood. To accompany our genome sequencing work of B. bassiana strain ARSEF 2860, fungal transcriptional responses to different niches were studied using an Illumina RNA_seq technique. To examine fungal response to insect cuticle, conidia were inoculated on locust hind wings for 24 hours before used for RNA extraction. To evaluate fungal adaptation to insect hemocole, the fifth instar larvae of cotton bollworms were injected with spore suspension and fungal cells isolated by centrifugation in a step gradient buffer. To unveil the mechanism of interaction with plants, the fungus was grown in corn root exudates for 24 hours. After RNA sequencing, around three million tags were acquired for each sample and fungal transcriptional profiles were compared.

overall_design: Unveiling gene differential expression patterns when the insect biocontrol fungus Beauveria bassiana grown in insect hemocoel, corn root exudates and on insect cuticles.

first: Peng

last: Zheng


title: Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses

status: Public on Jul 08 2012

city: Stanford

first: Eunkyoo

last: Oh

address: 260 Panama Street

department: Plant Biology

institute: Carnegie Institution

first: Zhiyong


title: Strand-specific ChIP-seq profiling of Rad52 localization

status: Public on Jul 07 2012

city: Beijing

first: Zhi-Xiong

last: Zhou

address: No.7, ZGC Life Science Park

institute: National Institute of Biological Sciences, Beijing

summary: We report the development of a novel strand-specific ChIP-seq strategy and application of this strategy in studying genome instability events

overall_design: We examined vulnerable genomic sites by applying strand-specific ChIP-seq of Rad52 to S. pombe haploid mutants showing elevated level of genome instability.

first: Mei-Jun

last: Zhang

first: Xu

last: Peng

first: Li-Lin

last: Du


title: Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses [ChIP-seq]

summary: Plant growth is coordinately regulated by environmental and hormonal signals. Brassinosteroid (BR) plays essential roles in growth regulation by light and temperature, but the interactions between BR and these environmental signals remain poorly understood at the molecular level. Here, we show that direct interaction between the dark- and heat-activated transcription factor phytochrome-interacting factor4 (PIF4) and the BR-activated transcription factor BZR1 integrates the hormonal and environmental signals. BZR1 and PIF4 interact with each other in vitro and in vivo, bind to nearly two thousand common target genes, and synergistically regulate many of these target genes, including the PRE family HLH factors required for promoting cell elongation. Genetic analysis indicates that BZR1 and PIFs are interdependent in promoting cell elongation in response to BR, darkness, or heat. These results show that the BZR1-PIF4 interaction controls a core transcription network, allowing plant growth co-regulation by the steroid and environmental signals.

overall_design: Genome-wide identification of PIF4 binding sites in etiolated Arabidopsis seedlings.


title: Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses [RNA-seq]

overall_design: RNA-Seq for Col-0, bzr1-1D, pifq and pifq;bzr1-1D seedlings grown on BRZ-containing medium in the dark.


title: RNA-seq from ENCODE/Caltech

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (mailto:georgi@caltech.edu for data coordination/informatics/experimental questions, mailto:diane@caltech.edu for informatics questions, mailto:bawilli_91125@yahoo.com for experimental questions). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

This track is produced as part of the ENCODE Project. RNA-seq is a method for mapping and quantifying the transcriptome of any organism that has a genomic DNA sequence assembly. RNA-seq is performed by reverse-transcribing an RNA sample into cDNA, followed by high throughput DNA sequencing, which was done here on an Illumina Genome Analyzer (GAI or GAIIx) (Mortazavi et al., 2008). The transcriptome measurements shown on these tracks were performed on polyA selected RNA (http://genome.ucsc.edu/cgi-bin/hgEncodeVocab?term=longPolyA&type=rnaExtract) from total cellular RNA (http://genome.ucsc.edu/cgi-bin/hgEncodeVocab?term=cell&type=localization) using two different protocols - one that preserves information about which strand the read is coming from and one that does not. Due to the specifics of the enzymology of library construction, gene and transcript quantification is more accurate based on the non-strand-specific protocol, while the strand-specific protocol is useful for assigning strandedness, but in general less reliable for quantification.
Non-strand-specific protocol (deep "reference" transcriptome measurements, 2x75 bp reads): PolyA-selected RNA was fragmented by magnesium-catalyzed hydrolysis and then converted into cDNA by random priming and amplified. Data have been produced in two formats: single reads, each of which comes from one end of a cDNA molecule, and paired-end reads, which are obtained as pairs from both ends of cDNAs. This RNA-seq protocol does not specify the coding strand. As a result, there will be ambiguity at loci where both strands are transcribed. The "randomly primed" reverse transcription is, apparently, not fully random. This is inferred from a sequence bias in the first residues of the read population, and this likely contributes to observed unevenness in sequence coverage across transcripts.
Strand specific protocol (1x75 bp reads): PolyA-selected RNA was fragmented by magnesium-catalyzed hydrolysis. 3' adapters were ligated to the 3' end of fragments, then 5' adapters were ligated to the 5' end. The resulting RNA molecules were converted to cDNA and amplified. This RNA-seq protocol does specify the coding strand as each read is in the same 5'-3' orientation as the original RNA strand. As a result, loci where both strands are transcribed can be disambiguated. However, RNA ligation is an inherently biased process and as a result greater unevenness in sequence coverage across transcripts is observed compared to the non-strand-specific data, and quantification is less accurate.
Data Analysis: Reads were aligned to the hg19 human reference genome using TopHat, a program specifically designed to align RNA-seq reads and discover splice junctions de novo. Cufflinks, a de novo transcript assembly and quantification software package, was run on the TopHat alignments to discover and quantify novel transcripts and to obtain transcript expression estimates based on the GENCODE annotation. All sequence files, alignments, gene and transcript models and expression estimates files are available for download.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
Experimental Procedures: Cells were grown according to the approved ENCODE cell culture protocols except for H1-hESC for which frozen cell pellets were purchased from Cellular Dynamics. Cells were lysed in RLT buffer (Qiagen RNEasy kit) and processed on RNEasy midi columns according to the manufacturer's protocol, with the inclusion of the "on-column" DNAse digestion step to remove residual genomic DNA.
75 µgs of total RNA was selected twice with oligo-dT beads (Dynal) according to the manufacturer's protocol to isolate mRNA from each of the preparations. For 2x75 bp non-stranded RNA-seq, 100 ngs of mRNA was then processed according to the protocol in Mortazavi et al (2008), and prepared for sequencing on the Genome Analyzer flow cell according to the protocol for the ChIPSeq DNA genomic DNA kit (Illumina). The majority of paired-end libraries were size-selected around 200 bp (fragment length) with the exception of a few additional replicates that were size-selected at 400 bp with the specific intent to investigate the effect of fragment length on results. Strand-specific RNA-seq libraries were prepared from 100ng of mRNA from the same preparation following Illumina's Strand-Specific RNA-seq protocol .
Libraries were sequenced with an Illumina Genome Analyzer I or an Illumina Genome Analyzer IIx according to the manufacturer's recommendations. Reads of 75 bp length were obtained, single end for directional, strand-specific libraries (1x75D) and paired end for non-strand-specific libraries (2x75).
Data Processing and Analysis: Reads were mapped to the reference human genome (version hg19), with or without the Y chromosome, depending on the sex of the cell line, and without the random chromosomes and haplotypes in all cases, using TopHat (version 1.0.14). TopHat was used with default settings with the exception of specifying an empirically determined mean inner-mate distance. After mapping reads to the genome and identifying splice junctions, the data was further analyzed using the transcript assembly and quantification software Cufflinks (version 0.9.3) using the sequence bias detection and correction option. Cufflinks was used in two modes: first, expression for genes and individual transcripts was quantified based on the GENCODE annotation, for both versions v3c and v4 of GENCODE GRCh37, and second, Cufflinks was run in de novo transcript assembly and quantification mode to obtain candidate novel transcript and gene models and expression estimates for them.

first: Ali

last: Mortazavi

first: Brian

last: Williams

first: Georgi

last: Marinov

first: Diane

last: Trout

first: Brandon

last: King

first: Ken

last: McCue

first: Lorian

last: Schaeffer

first: Norma

last: Neff

first: Florencia

last: Pauli

first: Fan

first: Tim

last: Reddy

first: Rami

last: Rauch

first: Chris

last: Partridge

first: Gary

last: Schroth

first: Shujun

last: Luo

first: Eric

last: Vermaas

first: Cole

last: Trapnell

first: Lior

last: Pachter

first: Steven

last: Salzberg


title: Control of Pro-Inflammatory Gene Programs by Regulated Trimethylation and Demethylation of Histone H4K20

status: Public on Jul 05 2012

city: La Jolla

first: Joshua

last: Stender

address: 9500 Gillman

institute: University of California at San Diego

summary: Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of co-regulator complexes that function to read, write and erase specific histone modifications linked to transcriptional activation or repression.  Here, we provide evidence for an unexpected role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR co-repressor complexes.  Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-KB-dependent delivery of the histone demethylase PHF2.  Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 tri-methylation/de-methylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.

overall_design: mRNA profiling from thioglycollate-elicited mouse macrophages treated with siRNA for Control, Smyd5 and Phf2 for 48 hours followed by 4 hours of LPS treatment.

first: Christopher

last: Glass


title: BEAF-32 insulator regulates gene expression of genes involved in cell polarity and neoplastic growth.

status: Public on Jul 06 2012

city: Atlanta

first: Gurudatta

last: Baraka Vishwanathan

state: GA

address: 1510 clifton road ne

department: Biology Department

institute: Emory University

first: Victor

last: Corces

last: Vishwanathan

first: Edward

last: Ramos


title: Genome binding profile of the conserved CSB-PGBD3 fusion protein in CSB-null UVSS1KO cells

city: Seattle

first: Lucas

last: Gray

state: WA

address: 615 Westlake Ave N

department: Molecular Genetics

institute: Allen Institute for Brain Science

summary: The CSB-PGBD3 fusion protein arose over 43 million years ago when a 2.5 kb piggyBac 3 (PGBD3) transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB) gene in the common ancestor of all higher primates. The CSB-PGBD3 fusion protein binds internally-deleted PGBD3 elements called MER85s in vitro, and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein.

overall_design: 1 ChIP sample and 1 unenriched input control from the same crosslinked chromatin pool

first: Alan

middle: M

last: Weiner

first: Jay

last: Shendure

first: Choli

last: Lee


title: UNG shapes the specifity of AID-induced somatic hypermutation in B cells

city: Madrid

first: Pablo

last: Perez-Duran

address: Melchor Fernandez Almagro, 3

country: Spain

institute: Centro Nacional de Investigaciones Cardiovasculares (CNIC)

summary: Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.

overall_design: Next Generation Sequencing analysis of mutations introduced by AID in activated B lymphocytes from WT and UNG-/- mice (n=4). Activated B cells from AID-/- mice (n=2) were used as negative controls.

last: Pérez-Durán

first: Almudena

middle: R

last: Ramiro


title: RNA-seq data of si-Snai1, si-Snai2, si-Snai1/2 and si-Scrambled treated mouse primary myoblasts

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

country: Canada

institute: Ottawa Hospital Research Institute

summary: In skeletal myogenesis, the transcription factor MyoD, activates distinct transcriptional programs in progenitors compared to terminally differentiated cells. Using ChIP-seq and gene expression analyses, we show that in primary myoblasts, Snai1/2-HDAC1/2 repressive complex bind and exclude MyoD from its targets. Notably, Snail binds E-box motifs that are G/C-rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation. By contrast, Snai1/2 does not bind the A/T-rich E-boxes associated with MyoD targets in myoblasts. Thus, Snai1/2-HDAC1/2 prevents MyoD occupancy on differentiation-specific regulatory elements and the change from Snail1/2- to MyoD-binding often results in enhancer switching during differentiation. Furthermore, we show that a regulatory network involving Myogenic Regulatory Factors (MRFs), Snail/2, miR-30a and miR-206 acts as a molecular switch that controls entry into myogenic differentiation. Together, these results reveal a regulatory paradigm that directs distinct gene expression programs in progenitors versus terminally differentiated cells.

overall_design: In vivo depletion of transcription factor with siRNA followed by whole transcriptome analysis (RNA-seq) to identify target genes.

first: Vahab

last: Soleimani

first: Michael

last: Rudnicki


title: Mapping regulatory elements using signatures of open chromatin in Arabidopsis thaliana

city: Madison

first: Yufeng

last: Wu

state: WI

address: 1575 Linden Drive

institute: UW-Madison

summary: Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements.  One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion.  We reported genome-wide high resolution maps of DNase I hypersensitive (DH) sites from both seedling and flower tissues of Arabidopsis from the Columbia (Col) ecotype and the corresponding ddm1 (deficient in DNA methylation 1) mutant. We identified 38,290, 41,193, 38,313, and 38,153 DH sites in leaf (Col), flower (Col), ddm1 leaf, and ddm1 flower tissues, respectively. Approximately 45% of the DH sites in all tissue types were located within 1 kb of a transcription start site (TSS), which represents a putative promoter region. Pairwise comparisons of the DH sites derived from different tissue types revealed DH sites specific to each tissue. DH sites are significantly associated with long non-coding RNAs (lncRNAs) and conserved non-coding sequences (CNSs). The binding sites of MADS-domain transcription factors AP1 and SEP3 are highly correlated with DH sites.

overall_design: To map the DH sites in A. thaliana, we constructed a total of five DNase-seq libraries using leaf and flower tissues from the Columbia (Col) ecotype and a ddm1 (deficient in DNA methylation 1) mutant of Columbia.  These libraries were sequenced using the Illumina Genome Analyzer.  We obtained a total of 190 million (M) sequence reads from these libraries.  Approximately 114 M reads had a single sequence match in the A. thaliana genome

first: Wenli

last: Zhang

first: Tao

first: Jiming

last: Jiang


title: UNG shapes the specificity of AID-induced somatic hypermutation


title: HDAC3 requirement for the inflammatory gene expression program in macrophages [ChIP_Seq]

first: Iros

last: Barozzi

summary: Pan-Hdac inhibitors (HDACi) are endowed with a potent anti-inflammatory activity, but the relative role of each of the eleven Hdac proteins sensitive to HDACi to the inflammatory gene expression program is unknown. Using an integrated genomic approach we found that Hdac3-deficient macrophages are unable to activate almost half of the inflammatory gene expression program when stimulated with lipopolysaccharide (LPS). A large part of the activation defect is due to loss of basal and LPS-inducible expression of IFNb, which in basal cells maintains Stat1 protein levels, and after stimulation acts in an autocrine/paracrine manner to promote a secondary wave of Stat1-dependent gene expression. We show that loss of Hdac3-mediated repression of nuclear receptors leads to hyperacetylation of thousands of genomic sites and associated gene derepression. The upregulation of the constitutively expressed prostaglandin endoperoxide synthase, Ptgs1 (Cox-1), has a causative role in the phenotype, since its chemical inhibition reverts the Ifnb activation defect. These data may have relevance for the use of selective Hdac inhibitors as anti-inflammatory agents.

overall_design: Chromatin immuno-precipitations of H4 histone pan-acetylation followed by multiparallel sequencing performed in murine bone marrow-derive macrophages. Experiments carried out in untreated cells as well as in cells treated for 4hrs with lipopolysaccharide (LPS), for both HDAC3 +/- (wt) and HDAC3 -/- (KO) mice.

first: Xuefen

last: Chen

first: Alberto

last: Termanini

first: Elena

last: Prosperini

first: Flore

last: Mietton

first: Gianluca

last: Matteoli

first: Scott

last: Hiebert

first: Gioacchino

last: Natoli


title: Independence of Repressive Histone Marks and Chromatin Compaction during Senescent Heterochromatic Layer Formation

city: Cambridge

first: Rafik

last: Salama


title: Distribution of Drosophila insulator protein BEAF-32 in Wing imaginal tissue (Wildtype) [ChIP-seq]

first: Gurudatta

last: Baraka Vishwanathan

summary: Chromatin insulators are DNA protein complexes that mediate inter and intra chromosomal interactions. Boundary Element Associated Factor- 32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 647 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins such as Bazooka, a determinant of cell polarity; the Insulin receptor-1 and the p70-S6 kinase, involved in the insulin growth factor pathway that can activate various pathways involved in cell proliferation; and the Unpaired 3 ligand of the Janus kinase (JNK) pathway, and its target gene Socs35, which can also activate cell proliferation. The expression of genes involved in DNA homologous end repair and oxidative stress pathways is also increased in BEAF-32 mutants. Among the down-regulated genes are those encoding components of the Wingless pathway, which is required for cell differentiation, and amino acid metabolism. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.

overall_design: Here we examine mapping the genomic binding sites and its distribution of BEAF-32, a Drosophila insulator proteins  in wing imaginal tissue. We performed ChIP-seq analysis using BEAF-32B.

first: Victor

last: Corces

last: Vishwanathan

first: Edward

last: Ramos


title: Transcriptomes of germinal zones of human and mouse fetal neocortex suggest a role of extracellular matrix in progenitor self-renewal.

status: Public on Jul 03 2012

city: Leipzig

first: Martin

last: Kircher

address: Deutscher Platz 6

institute: Max Planck Society

summary: The expansion of the neocortex during mammalian brain evolution results primarily from an increase in neural progenitor cell divisions in its two principal germinal zones during development, the ventricular zone (VZ) and the subventricular zone (SVZ). Using mRNA sequencing, we analyzed the transcriptomes of fetal human and embryonic mouse VZ, SVZ and cortical plate (CP). We describe sets of genes that are up- or down-regulated in each germinal zone. These data suggest that cell adhesion and cell-extracellular matrix (ECM) interactions promote the proliferation and self-renewal of neural progenitors in the developing human neocortex. Notably, relevant ECM-associated genes include distinct sets of collagens, laminins, proteoglycans and integrins, along with specific sets of growth factors and morphogens. Our data establish a basis for identifying novel cell-type markers and open up avenues to unravel the molecular basis of neocortex expansion during evolution.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Total RNA was isolated from the VZ, inner SVZ (ISVZ), outer SVZ (OSVZ) and CP of six 13-16 weeks post-conception (w.p.c.) human fetuses and from the VZ, SVZ and CP of five E14.5 mouse embryos using laser capture microdissection of Nissl-stained cryosections of dorsolateral telencephalon. Poly A+ RNA was used as template for the preparation of cDNA which were then subjected to single-end 76-bp RNA-Seq.

first: Simone

middle: A

last: Fietz

first: Wieland

last: Huttner

first: Svante

last: Pääbo


title: Roles of RDR701 and DCLs in Rice PhasiRNA biogenesis

first: xiaofeng

last: cao

address: West Lincui Road, Chaoyang District

department: State Key Laboratory of Plant Genomics

institute: Institute of Genetics and Developmental Biology

summary: In this study, we analyzed function of the rice RDR701 and various DCLs proteins in small RNA processing. small RNA deep sequencing analyses revealed that RDR701 and OsDCL4 is involved in tasiRNAs and 21- and 24-nt phasiRNAs biogenesis. Further analyses indicate RDR701 coupling with OsDCL4 and OsDCL3b are required for the biogenesis of 21- and 24-nt phasiRNAs, respectively. The triggering miRNAs for intial both size phasiRNAs,are also shown RDR701- and OsDCL4-dependent, suggesting two sequential round activity of RDR701 in associated with different DCLs are essential for rice phasiRNAs biogenesis.

overall_design: Four small RNA libraries were constructed from panicles of zhonghua11 (a wild-type Japonica rice variety) and rdr701-1 at high temperature (Day average temperature 32℃) and low temperature (Day average temperature 28℃).

first: Xianwei

last: Song

first: Pingchuan

last: Li

first: Dekai

last: Wang

first: Lijia

last: Ma

first: Jixian

last: Zhai

first: Ming

last: Zhou

first: Bin

last: Liu

first: Dong-Hoon

last: Jeong

first: Shouyun

last: Cao

first: Xia

last: Cui

first: Chunyan

first: Chengcai

last: Chu

first: Xiu-Jie

first: Xuemei

first: Pamela

middle: J

last: Green

first: Blake

last: Meyers

first: Yuezhi

last: Tao

first: Xiaofeng


title: Shutdown is a component of the Drosophila piRNA biogenesis machinery

status: Public on Jul 01 2012

city: Cold Spring Harbor

first: Benjamin

last: Czech

address: 1 Bungtown Rd

institute: Cold Spring Harbor Laboratory


title: Shutdown is a component of the Drosophila piRNA biogenesis machinery (smallRNA)

summary: In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping- pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germ line and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins, with domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones. Though the relevance of these domains to piRNA biogenesis is unknown, evolutionary comparisons raise questions about the integrity of these functions in the shutdown protein.

overall_design: Examination of small RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of white, shu and piwi in ovaries of Drosophila melanogaster by deep sequencing (using Illumina GAII).

first: Jonathan

middle: B

last: Preall

first: Paloma

last: Guzzardo

first: Felix

last: Muerdter

first: Gregory

last: Hannon


title: Shutdown is a component of the Drosophila piRNA biogenesis machinery (RNA-seq)

summary: In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure.  Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway.  Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle.  shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.

overall_design: Analysis of mRNA expression in Drosophila OSS cells transfected with GFP dsRNA.  One sample and replicate, used to establish the OSS baseline transcriptome in the presence of exogenous RNAi activity.


title: Genome-wide maps of nucleosome positions in Saccharomyces strains bearing heterologous DNA on yeast artificial chromosomes

city: Worcester

first: Amanda

last: Hughes

address: 364 Plantation Street

institute: University of Massachusetts Medical School

summary: We identify the cis and trans determinants of nucleosome positioning using a functional evolutionary approach involving S. cerevisiae strains containing large genomic regions from other yeast species. In a foreign species, nucleosome depletion at promoters is maintained over poly(dA:dT) tracts, whereas internucleosome spacing and all other aspects of nucleosome positioning tested are not. Interestingly, the locations of the +1 nucleosome and RNA start sites shift in concert. Strikingly, in a foreign species, nucleosome-depleted regions occur fortuitously in coding regions, and they often act as promoters that are associated with a positioned nucleosome array linked to the length of the transcription unit.

overall_design: nucleosome mapping for 3 strains bearing yeast artificial chromosomes from Kluyveromyces lactis and 2 strains with Debaryomyces hansenii artificial chromosomes in Saccharomyces cerevisiae

first: Oliver

last: Rando

first: Kevin

last: Struhl

first: Yi

last: Jin


title: Identification of new microRNAs in paired normal and tumor breast tissue suggests a dual role for the ERBB2/Her2 gene

city: Lund

first: Helena

last: Persson

address: Scheelevagen 2

department: Dept of Clinical Sciences, Oncology

country: Sweden

institute: Lund University

summary: To comprehensively characterize microRNA (miRNA) expression in breast cancer, we performed the first extensive next-generation sequencing expression analysis of this disease. We sequenced small RNA from tumors with paired samples of normal and tumor-adjacent breast tissue. Our results indicate that tumor identity is achieved mainly by variation in the expression levels of a common set of miRNAs rather than by tissue-specific expression. We also report 361 new, well-supported miRNA precursors. Nearly two-thirds of these new genes were detected in other human tissues and 49% of the miRNAs were found associated with Ago2 in MCF7 cells. Ten percent of the new miRNAs are located in regions with high-level genomic amplifications in breast cancer. A new miRNA is encoded within the ERBB2/Her2 gene and amplification of this gene leads to overexpression of the new miRNA, indicating that this potent oncogene and important clinical marker may have two different biological functions. In summary, our work substantially expands the number of known miRNAs and highlights the complexity of small RNA expression in breast cancer.

overall_design: Sequencing of approximately 18-35 nt small RNAs from paired samples of normal, tumor and tumor-adjacent tissue for five breast cancer patients

first: Anders

last: Kvist

first: Natalia

last: Rego

first: Johan

last: Staaf

last: Vallon-Christersson

first: Lena

last: Luts

first: Niklas

last: Loman

first: Göran

last: Jönsson

first: Hugo

last: Naya

first: Mattias

last: Höglund

first: Ake

last: Borg

first: Carlos

last: Rovira


title: CpG Island Structure and Trithorax/Polycomb Chromatin Domains in Human Cells

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: TrxG and PcG complexes play key roles in the epigenetic regulation of development through H3K4me3 and H3K27me3 modification at specific sites throughout the human genome, but how these sites are selected is poorly understood. We find that in pluripotent cells, clustered CpG islands at genes predict occupancy of H3K4me3 and H3K27me3, and these "bivalent" chromatin domains precisely span the boundaries of CpG island clusters.

overall_design: Examination of two histone modifications (H3K4me3 and H3K27me3) in human induced pluripotent stem (hiPS) M23F cells.

first: Matthew

last: Guenther


title: miRNA sensor and decoy libraries permit high-throughput assessment of miRNA activity and function

city: New York

first: Brian

last: Brown

address: 1470 Madison Avenue

department: Genetics and Genomic Sciences

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: mRNA profiling of THP1 cell line

summary: Analysis of mRNA in THP1 (human monocytic leukemia) cell line in order to correlate miRNA activity with target abundance.

overall_design: THP1 mRNA profiles were generated in triplicates by deep-sequencing in Illumina HiSeq2000.

middle: D

first: Ravi

last: Sachidanandam


title: MCMV infection of cultured mouse cells induces expression of miR-7a.

city: Munich

first: Klaus

last: Förstemann

address: Feodor-Lynen 25

department: Gene Center

institute: Ludwig-Maximilians University

summary: One goal of viral infection is to reprogram the host cell to optimize viral replication. As part of this process, viral miRNAs may compete for components of the miRNA/siRNA pathway as well as regulate cellular targets. Mouse Cytomegalovirus has been described to generate large numbers of viral miRNAs during lytic infection and was therefore used to analyze the impact of viral miRNAs on the host cell small RNA system as well as to check for sorting of viral small RNAs into specific Ago-proteins. Deep sequencing analysis of MCMV infected cells revealed that viral miRNAs represent only app. 13% of all detected miRNAs. All previously described MCMV miRNAs with the exception of miR-m88-1* were confirmed and for the MCMV miR-m01-1 hairpin an additional miRNA, designated miR-m01-1-3p, was found. Its presence was confirmed by qPCR and Northern Blot. Deep sequencing after RISC IP with antibodies specific for either Ago1 or Ago2 showed that all MCMV miRNAs are loaded into both RISC complexes. The ratio of MCMV to mouse miRNAs was not increased after immunoprecipitation of Ago-proteins. Viral miRNAs therefore do not overwhelm the host miRNA processing system nor are they preferentially incorporated into RISC. We found that 3 mouse miRNAs showed an altered expression due to MCMV infection. Down-regulation of miR-27a, as previously described, could be confirmed. In addition, miR-26a was down-regulated and an up-regulation of miR-7a dependent on viral protein expression could be observed.

overall_design: Examination of small RNA expression in uninfected vs. infected cells, immunoprecipitation + sequencing of Ago1 and Ago2 loaded small RNAs in infected cells

first: Alexandra

last: Dittmer


title: UNG shapes the specifity of AID-induced somatic hypermutation in non B cells

first: Pablo

last: Perez-Duran

overall_design: Next Generation Sequencing analysis of mutations introduced by AID in non B cells. NIH-3T3 cells were co-transduced with mOrangeSTOP and AID-ER–expressing vectors, together with Ugi (UNG inhibitor), UNG, or empty vector as control (n=3). Transduced cells were cultured in the presence of OHT during 11 d. AID-E58Q-ER vector (catalytically inactive form of AID) was used as a negative control in combination with the previously described constructions (n=3).

last: Pérez-Durán

first: Laura

last: Belver

first: Virginia

last: de Yébenes

first: Pilar

last: Delgado

first: David

last: Pisano

first: Almudena

last: Ramiro


title: A draft map of cis-regulatory sequences in the mouse genome [HiC]

first: Bing

last: Ren

state: CA

address: 9500 Gilman Drive

institute: University of California, San Diego

summary: As the most widely used mammalian model organism, mice play a critical role in biomedical research for mechanistic study of human development and diseases. Today, functional sequences in the mouse genome are still poorly annotated a decade after its initial sequencing. We report here a map of nearly 300,000 cis-regulatory sequences in the mouse genome, representing active promoters, enhancers and CTCF binding sites in a diverse set of 19 tissues and cell types. This map provides functional annotation to nearly 11% of the genome, and over 70% of conserved, non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we demonstrate that cis-regulatory sequences are organized into domains of coordinately regulated enhancers and promoters. Our results provide a valuable resource for the annotation of functional elements in the mammalian genome, and study of regulatory mechanisms for tissue-specific gene expression.

overall_design: Cortex Hi-C experiment were conducted in biological replicates

first: Yin

last: Shen

first: Feng

last: Yue


title: Nucleotidyltransferase gene HESO1

city: shenzhen

first: Lei

last: Gao

state: guangdong

address: Nanhai Ave 3688

department: College of Life Sciences

institute: shenzhen university

summary: Small RNAs in plants are protected by 2'-O-methylation at 3' terminal nucleotide by HEN1. In hen1 mutant, small RNAs lacking 3' methylation are truncated and tailed at 3' end. We identified a nucleotidyltransferase gene HESO1 in Arabidopsis that tails small RNAs in hen1. In order to study the small RNA tailing and truncation in detail in wild type Col, heso1-1, hen1-8 and hen1-8 heso1-1 double mutant, we constructed small RNA libraries for these four genotypes and we found heso1-1 mutation reduces small RNA tailing in hen1-8 background, however, the 3' truncation is not significantly affected.

overall_design: 8 samples examined: wild-type Col (replicate 1), heso1-1 (replicate 1), hen1-8(replicate 1), hen1-8 heso1-1 (replicate 1), wild-type Col (replicate 2), heso1-1 (replicate 2), hen1-8(replicate 2) and hen1-8 heso1-1 (replicate 2).

first: Y

last: Zhao

last: Yu

first: B

last: Mo

first: X

last: Chen


title: Unraveling cell type–specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs

city: MONTPELLIER

first: Emilie

last: Besnard

address: 141 rue de la Cardonille

country: France

institute: Institute of Functional Genomics

summary:
Short nascent strands purification coupled to next-generation sequencing allowed us to identify replication origins on human genome in an extensive way, by mapping replication origins in 4 different cell types, IMR-90 fibroblasts, hESC H9 cells, iPSC Th Cl-4 cells and HeLa cells.
We demonstrated the existence of a cell type-specific reprogrammable signature of the cell identity revealed by specific efficiencies of conserved origin positions and not by the selection of cell-type specific subsets of origins.

overall_design: 4 different cell types were analyzed. For each cell types, 2 different biological replicates of short nascent strands at replication origins were purified. Each SNS sample was sequencing at least one time.

first: Amélie

last: Babled

first: Laure

last: Lapasset

first: Ollivier

last: Milhavet

first: Hugues

last: Parrinello

first: Christelle

last: Dantec

first: Jean-Michel

last: Marin

first: Jean-Marc

last: Lemaitre


title: microRNA profile of human nasopharyngeal tissues: nasopharyngeal carcinoma tissues vs. normal nasopharyngeal tissues

city: Guangzhou

first: Jun

last: Ma

address: 651 Dongfeng Road East

institute: Sun Yat-sen University Cancer Center

summary: MicroRNAs are biomarkers of prognosis and survival for many types of cancer.  We evaluated whether microRNAs can predict the survival and efficacy of concurrent chemotherapy in nasopharyngeal carcinoma (NPC) patients. We retrospectively analyzed microRNA expression in 312 paraffin-embedded NPC specimens and 18 normal nasopharyngeal tissues using microarray. We found Forty-one microRNAs are differentially expressed between NPC and normal tissues, and a five-microRNA signature can predict survival independent of stage. NPC patients with the low-risk microRNA signature have a favorable response to concurrent chemotherapy.

overall_design:
microRNA profiling of  nasopharyngeal carcinoma tissues vs. normal nasopharyngeal tissues
312 paraffin-embedded nasopharyngeal carcinoma tissues and 18 paraffin-embedded normal nasopharyngeal tissues

first: Huiyun

last: Wang

last: Liu


title: ChIP-Seq of Myf5, MyoD, Snai1, HDAC1, HDAC2, E47 and empty vector controls in mouse skeletal myoblasts or myotubes

first: Theodore

last: Perkins

summary: In skeletal myogenesis, the transcription factor MyoD activates distinct transcriptional programs in progenitors compared to terminally differentiated cells. Using ChIP-seq and gene expression analyses, we show that in primary myoblasts, Snail-HDAC1/2 repressive complex bind and exclude MyoD from its targets. Notably, Snail binds E-box motifs that are G/C-rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation. By contrast, Snail does not bind the A/T-rich E-boxes associated with MyoD targets in myoblasts. Thus, Snai1-HDAC1/2 prevents MyoD occupancy on differentiation-specific regulatory elements and the change from Snail- to MyoD-binding often results in enhancer switching during differentiation. Furthermore, we show that a regulatory network involving Myogenic Regulatory Factors (MRFs), Snail/2, miR-30a and miR-206 acts as a molecular switch that controls entry into myogenic differentiation. Together, these results reveal a regulatory paradigm that directs distinct gene expression programs in progenitors versus terminally differentiated cells.

overall_design: Genome wide binding sites of various transcription factors and chromatin modifiers in muscle cells

first: Vahab

last: Soleimani

first: Michael

last: Rudnicki


title: A draft map of cis-regulatory sequences in the mouse genome [ChIP-Seq]

first: Bing

last: Ren

summary: As the most widely used mammalian model organism, mice play a critical role in biomedical research for mechanistic study of human development and diseases. Today, functional sequences in the mouse genome are still poorly annotated a decade after its initial sequencing.  We report here a map of nearly 300,000 cis-regulatory sequences in the mouse genome, representing active promoters, enhancers and CTCF binding sites in a diverse set of 19 tissues and cell types. This map provides functional annotation to nearly 11% of the genome, and over 70% of conserved, non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we demonstrate that cis-regulatory sequences are organized into domains of coordinately regulated enhancers and promoters. Our results provide a valuable resource for the annotation of functional elements in the mammalian genome, and study of regulatory mechanisms for tissue-specific gene expression.

overall_design: 19 tissues and primary cell types were examined.

first: Yin

last: Shen

first: Feng

last: Yue


title: A draft map of cis-regulatory sequences in the mouse genome [RNA-Seq]


title: Parallel high-throughput RNA-sequencing suggests little overlap of differential gene expression between knockout of TDP-43 and its over-expression in central nervous system in Drosophila

city: Portland

first: Dennis

last: Hazelett

state: OR

address: 611 SW Campus Dr

department: Integrative Biosciences

institute: OHSU

summary: The human Tar-DNA-binding protein TDP-43 is closely associated with ALS and other neurodegenerative disorders. TDP-43 contains two highly conserved RNA-binding motifs and possesses a variety of documented roles in RNA metabolism, including pre-RNA splicing and repression of transcription. We sought to measure the effect that knockout and over-expression of the fly orthologue of this protein, Tar-DNA-binding protein homolog (TBPH), has on the transcriptome of the central nervous system (CNS) of Drosophila melanogaster. To this end, we used massively parallel sequencing methods (RNA-seq) to transcriptionally profile the CNS in loss-of-function mutants and gain-of-function over-expression genotypes. We found that loss of TBPH resulted in widespread gene activation, much of which could be reversed by rescue of TBPH expression, suggesting that repression is one of the major roles of TBPH. Conversely, we found that over-expression of TBPH resulted largely in decreased gene expression. However, there was little overlap in the genes which were affected in these two genotypes, suggesting that the bulk of genes affected by TBPH loss-of-function and over-expression are different. We provide a comprehensive look at enriched gene ontologies in both cases, suggesting that TDP-43 plays a role in regulating basic processes in neurons. We also describe a number of genes whose splicing is likely to be altered in the absence of TDP-43.

overall_design:
In this study we compare the effects of knockout of the TDP-43 ortholog (TBPH) in the fly nervous system with the effects of its overexpression.

2 treatment groups are presented. In the first, TDP-43 knockout (G2) is compared to control (A1) and rescue (G2; TDP-43-GAL4>UAS-TDP-43). In the second comparison, the motor neuron driver D42-GAL4 is used to overexpress TDP-43 in motor neurons, using LacZ as a control for overexpression of a foreign protein.

first: David

last: Morton