title: The maize silks transcriptome

status: Public on Sep 14 2012

city: East Lansing

first: Erich

last: Grotewold

state: MI

address: 603 Wilson Road

institute: Michigan State University

summary: P1 encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize silks and red phlobaphene pigments in pericarps and other floral tissues, which contributed to making P1 an important visual marker since the dawn of modern genetics. We conducted RNA-Seq using from maize silks obtained at 2-3 days after emergence. High-throughput sequencing using the Illumina platform resulted in the generation of ~14 million high quality reads, corresponding to ~7 million reads for each sample, from which 76% aligned to the maize genome.

overall_design: Examination of two different RNA samples from maize silks obtained at 2-3 days after emergence

first: Maria

last: Casas

first: Kengo

last: Morohashi

last: Mejia-Guerra

first: Alper

last: Yilmaz


title: A transcription factor based mechanism for mouse heterochromatin formation

status: Public on Sep 16 2012

city: Freiburg im Breisgau

first: Thomas

last: Manke

address: Stuebeweg 51

department: Bioinformatics and Deep-Sequencing Unit

country: Germany

institute: Max-Planck-Institute for Immunobiology and Epigenetics

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We identify Pax3 and Pax9 transcription factors (TFs) as important regulators of mouse heterochromatin. Simultaneous depletion of these factors results in loss of H3K9me3 from Major Satellite repeats and dramatic transcript overexpression. H3K9me3 enrichment at intergenic major satellite repeats is only present if these sequences retain intact Pax and other TF binding sites.

overall_design: H3K9me3 ChIP-seq in mouse ES cells wt and Pax3 KO and Pax3 KO/Pax9 KD

first: Inti

middle: A

last: De La Rosa-Velazquez

first: Aydan

middle: B

last: Karslioglu

first: Sarah

last: Diehl

last: Jenuwein


title: Lactation-Related microRNA Expression Profiles of Porcine Breast Milk Exosomes

status: Public on Sep 17 2012

city: Ya'an

first: Mingzhou

last: Li

state: Sichuan

address: No.46  Xinkang Road

department: College of Animal Science and Technology

country: China

institute: Sichuan Agricultural University

type: Non-coding RNA profiling by high throughput sequencing

summary: Breast milk is the primary source of nutrition for newborns, and rich in immunological components. microRNAs (miRNAs), a well-defined group of non-coding small RNAs, are present in various body fluids (such as breast milk), which are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and regulate target gene expression and recipient cell function. We present the lactation-related miRNA expression profiles in porcine milk exosomes across entire lactation period in pig industry (newborn to 28 days after birth) using deep sequencing technology. We found that the immune-related miRNAs are presented and enriched in breast milk exosomes, and generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs exhibited the higher abundances in the colostrum (newborn to 3 days after birth) than that in the mature milk (7 to 28 days after birth), as well as in the serum of colostrum-feeding piglets compared with the only mature milk-feeding piglets. These immune-related miRNAs-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are prelude to the in-depth investigations of the essential roles of the breast milk in the development of the infant’s immune system.

overall_design: Eight small RNA libraries in porcine breast milk exosomes of six lactigenous stages (0, 3, 7, 14, 21 and 28 days after birth) from three female pigs were sequenced.

first: Yiren

last: Gu

first: Xuewei

first: Xuebin

last: Lv


title: Impact of STAT5 dosage and binding location on genetic and biological programs activated in mouse mammary epithelium during pregnancy.

status: Public on Sep 18 2012

city: Cheonan

first: Keunsoo

last: Kang

state: Chungnam

address: Dandae-ro 119

department: Microbiology

country: South Korea

institute: Dankook University

summary: The transcription factor STAT5 has long been recognized as an important mediator of prolactin induced mammary development during pregnancy. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts different target promoters.

overall_design: Examination of genome-wide STAT5A, STAT5B, PolII and H3K4me3 in wild type (AABB) and Stat5a-null (BB) mammary tissues.

first: Daisuke

last: Yamaji

first: Gertraud

middle: W

last: Robinson

first: Lothar

last: Hennighausen


title: DNaseI Hypersensitivity by Digital DNaseI from ENCODE/PSU

status: Public on Sep 13 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 (Ross Hardison mailto:rch8@psu.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).
Rationale for the Mouse ENCODE project: Knowledge of the function of genomic DNA sequences comes from three basic approaches. Genetics uses changes in behavior or structure of a cell or organism in response to changes in DNA sequence to infer function of the altered sequence. Biochemical approaches monitor states of histone modification, binding of specific transcription factors, accessibility to DNases and other epigenetic features along genomic DNA. In general, these features are associated with gene activity, but the precise relationships remain to be established. The third approach is evolutionary, using comparisons among homologous DNA sequences to find segments that are evolving more slowly or more rapidly than expected given the local rate of neutral change. Such changes are inferred to be under negative or positive selection, respectively, and interpreted as DNA sequences needed for a preserved (negative selection) or adaptive (positive selection) function.
The ENCODE project aims to discover all the DNA sequences associated with various epigenetic features, with the reasonable expectation that these will also be functional (best tested by genetic methods). However, it is not clear how to relate these results with those from evolutionary analyses. The mouse ENCODE project aims to make this connection explicitly and with a moderate breadth. Assays identical to those being used in the ENCODE project are performed in cell types in mouse that are similar or homologous to those studied in the human project. The comparison will be used to discover which epigenetic features are conserved between mouse and human, and examine the extent to which these overlap with the DNA sequences under negative selection. The contribution of functional DNA preserved in mammals versus function in only one species will be discovered. The results will have a significant impact on the understanding of the evolution of gene regulation.
Maps of DNaseI Sensitivity: DNaseI has long been used to map general chromatin accessibility, and DNaseI hypersensitivity is a universal feature of active cis-regulatory sequences. Maps of DNaseI sensitivity measured genome-wide are generated through DNaseI digestion, addition of linkers at the sites of cleavage, and library prep followed by massively parallel short read sequencing on the Illumina GAIIx and HiSeq platforms. The sequence tags are mapped back to the mouse genome, and a graph of the smoothed kernel density of DNaseI cleavage sites is displayed as the "Signal" track. This provides a quantitative estimate of the frequency of cleavage by DNaseI in the initial digest, which in turn is related to the accessibility of the DNA in the chromatin. Segments of greatest cleavage site density represent DNase hypersensitive sites (DHSs) and are identified as peaks by the F-seq program (Boyle et al. 2008). DHSs are candidates for any cis-regulatory module, including promoters, enhancers, insulators, and novel elements. The sequence reads, quality scores, and alignment coordinates from these experiments 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:
Cells were grown and harvested according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell/mouse) for G1E and G1E-ER4.
DNaseI hypersensitive sites were isolated using methods called DNase-seq or DNase-chip (Song and Crawford, 2010). Briefly, cells were lysed with NP40, and intact nuclei were digested with optimal levels of DNaseI enzyme. DNaseI-digested ends were captured from three different DNase concentrations, and material was sequenced using Illumina sequencing.
The read length for sequences from DNase-seq is 20 bases long due to a MmeI cutting step of the approximately 50 kb DNA fragments extracted after DNaseI digestion. Sequences from each experiment were mapped to the mouse genome (mm9 assembly) using the program Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) (Langmead et al., 2009). Reads mapping to more than one location were not removed. For such reads, only the best mapping result was used ("--best" option). Sequences from multiple lanes were combined for a single replicate and converted to the sam/bam format using SAMtools (http://samtools.sourceforge.net/). Using F-seq, the resulting digital signal was converted to a continuous wiggle track that employs a Parzen kernel density estimation to create base pair scores (Boyle et al., 2008).
Discrete DNaseI HS sites (peaks) were identified from the DNase-seq F-seq density signal. Significant regions were determined by fitting the data to a gamma distribution to calculate p-values.

first: Christine

last: Dorman

first: Greg

last: Crawford

first: Chris

last: Morrissey

first: Yoichiro

last: Shibata

first: Terry

last: Furey

first: Anton

last: Nekrutenko

first: James

last: Taylor

first: Ross

last: Hardison


title: Synchronization of flowering transition by the tomato TERMINATING FLOWER gene

city: Cold Spring Harbor

first: Ke

last: Jiang

state: NY

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

type: Expression profiling by high throughput sequencing

summary: We sequenced mRNA from late vegetative meristems of tomato mutant TERMINARING FLOWER as well as the wild type progenitor to see genome-wide expression changes affected by the mutation.

overall_design: Meristems from multiple plants at the same developmental stages were collected for both mutants and wild types. The meristems were pooled together for mRNA extractions. Two biological replicates for each genotypes were prepared for mRNA extraction and sequencing library construction.

first: Cora

last: MacAlister

first: Soon

middle: J

last: Park

first: Fabien

last: Marcel

first: Abdelhafid

last: Bendahmane

first: Yinon

last: Izkovich

first: Yuval

last: Eshed

first: Zachary

last: Lippman


title: The maize pericarp transcriptome

first: Erich

last: Grotewold

summary: P1 encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize silks and red phlobaphene pigments in pericarps and other floral tissues, which contributed to making P1 an important visual marker since the dawn of modern genetics. We conducted RNA-Seq using pericarps at two different stages, 14 and 25 days after pollination (DAP). High-throughput sequencing using the Illumina platform resulted in the generation of ~20 million high quality reads, from which ~90% aligned to the recently completed maize genome sequence corresponding to ~5 million reads for each one of the four samples.

overall_design: Examination of two different RNA samples from two different stages of maize pericarp tissues.

first: Kengo

last: Morohashi

first: Maria

middle: K

last: Mejia-Guerr

middle: I

last: Casas


title: ChIPseq analysis of IRF4 and BATF in immune cells

city: South San Francisco

first: Smita

last: Agrawal

address: 1 DNA Way

department: Immunology

institute: Genentech Inc.

summary: Purpose: The purpose of this study is to find the binding partner of IRF4 in the context of Th17- cell differentiation. To this end, we have used ChIPseq analysis followed by de novo motif search around genome-wide binding sites to identify BATF as the binding partner for IRF4 in the context of not only Th17 cells but other immune cell types as well.

overall_design: Naïve T-cells isolated from the spleen of C57BL/6J mice are cultured under Th17, Th2 or Th0 polarizing conditions for 42 hrs and subject to ChIP using IRF4 and/or BATF antibodies followed by high-throughput sequencing.  Bone marrow derived dendritic cells (BMDCs) were stimulated with LPS for 6hrs and similrly subjected to ChIPseq analysis with IRF4.

first: Wenwen

last: Zeng

first: Bryan

last: Lugt


title: Epigenomic profiling of enhancers identifies orphan nuclear receptors NR2F1 and NR2F2 as novel regulators of human neural crest.

first: Alvaro

last: Rada

address: 269 Campus Drive, CCSR Building

department: Chemical and Systems Biology

institute: Stanford University

summary: Combining an in vitro hNCC differentiation protocol with epigenomic profiling, we provide the first whole-genome characterization of cis-regulatory elements in this highly relevant cell type. With this data at hand, we have characterized the chromatin state and dynamics of all human gene promoters during the course of NCC in vitro differentiation. Most importantly, we have identified a large cohort of active and NCC-specific enhancers, which we showed to be functionally relevant in vivo, in the context of embryonic development. Finally, through sequence analysis of the identified NCC enhancers, we uncovered the orphan nuclear receptors NR2F1 and NR2F2 as novel hNCC transcriptional regulators both in vitro and in vivo.

overall_design: Genome-wide analysis of H3K27ac in chicken neural crest cells (NCC) obtained from stage 11-14 or stage 20 embryos

last: Rada-Iglesias

first: Joanna

last: Wysocka


title: Chromatin based modeling of transcription rates identifies the contribution of different regulatory layers to steady-state mRNA levels.

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary: Messenger RNA levels in eukaryotes are balanced by two consecutive regulatory layers. Primary, transcriptional regulation at the level of chromatin and secondary, post-transcriptional regulation of the initial transcript in the cytoplasm. Each layer is individually studied in mechanistic detail, while integration of both processes is required to quantify the individual contribution to steady-state RNA levels. Here we show that chromatin features are sufficient to model transcription rate but with different sensitivities in dividing versus post mitotic cells. In both cases chromatin derived transcript levels explains over 80% of variance in measured RNA level enabling to separate transcription from different post-transcriptional processes. By further inclusion of measurements of mRNA half-life and micro RNA expression data we identify a low quantitative contribution of RNA decay by either micro RNA or general differential turnover to final mRNA levels. Together this establishes a chromatin based quantitative model for the contribution of transcriptional and posttranscriptional processes to steady-state levels of messenger RNA.

overall_design: Strand specific expression profiling by high throughput sequencing.

first: Sylvia

middle: C

last: Tippmann

first: Robert

last: Ivanek

first: Dimos

last: Gaidatzis

first: Anne

last: Schöler

first: Erik

last: van Nimwegen

first: Peter

middle: F

last: Stadler

first: Michael

last: Schübeler


title: A genome wide methylation map of neuroblastoma cell lines

city: Ghent

first: Maté

last: Ongenaert

address: De Pintelaan 185 - MRB

department: Center for Medical Genetics

country: Belgium

institute: Ghent University

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Methylation map of 8 neuroblastoma cell lines: NGS after MBD2-capture

summary: 8 neuroblastoma (NB) cell lines (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) their methylome is determined by sequencing after MBD2-capture using MethylCollector (ActiveMotif)

overall_design: 8 NB cell lines were included (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) in this study. After shearing (fragments of about 200 bp), DNA was captured using MBD2-capture (MethylCollector - ActiveMotif) followed by library preparation and multiplexing. Captured sequence tags were sequenced paired-end (2 x 45 bp) on Illumina GAIIx.

first: Anneleen

last: Decock

first: MatÃ©

first: Jo

last: Vandesompele

first: Frank

last: Speleman


title: The P1 Transcription Factor Binding to the Genome

first: Erich

last: Grotewold

summary: To determine which of the genes differentially expressed between P1-rr and P1-ww pericarps were immediate (direct) targets of P1, we conducted chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) using P1 polyclonal antibodies (alphaP1344) that recognize the non-conserved C-terminal region of P1 (Falcone Ferreyra et al., 2010), on pericarp chromatin.

overall_design: Comparison of pericarp chromatin inmunoprecipitated material P-rr_14DAP (P1 expressed) vs P-ww_14DAP (P1 not expressed) to determine P1 direct targets

first: Kengo

last: Morohashi

first: Alper

last: Yilmaz

first: Maria

last: Mejia-Guerra


title: A genomic regulatory element that directs assembly and function of immunespecific AP-1-IRF complexes

first: Smita

last: Agrawal

first: Harinder

last: Singh


title: RNA-seq experiments in human neural crest cells (hNCC)

first: Alvaro

last: Rada

last: Rada-Iglesias

first: Joanna

last: Wysocka


title: Epigenomic profiling of enhancers identifies orphan nuclear receptors NR2F1 and NR2F2 as novel regulators of human neural crest


title: Genome-wide analysis of p300, H3K4me3, H3K4me1, H3K27me3 and H3K27ac in in vitro differentiated human neural crest cells (hNCC)

overall_design: Genome-wide analysis of p300, H3K4me3, H3K4me1, H3K27me3, H3K27ac, NR2F1, NR2F2 and TFAP2A in in vitro differentiated human neural crest cells (hNCC)


title: Genome-wide mapping of nucleosome positioning and DNA methylation within Individual DNA molecules

status: Public on Sep 12 2012

city: Chicagp

first: Yaping

last: Liu

state: IL

address: 303 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University

summary: DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions, that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule and thus, single cell level that can be used to monitor disease progression and response to therapy.

overall_design: Nucleosome Occupancy and Methylome-Sequencing (NOMe-Seq) on IMR90 cell line and 2 GBM cell lines

first: Liu

last: Yaping

first: Berman

middle: P

last: Benjamin

first: Kelly

last: Terry


title: Whole Genome Bisulfite Sequencing by ENCODE/HAIB

first: ENCODE

last: DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (). 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 reports the percentage of DNA molecules that exhibit cytosine methylation. In general, DNA methylation within a gene's promoter is associated with gene silencing, and DNA methylation within the exons and introns of a gene is associated with gene expression. Proper regulation of DNA methylation is essential during development and aberrant DNA methylation is a hallmark of cancer. DNA methylation status was assayed with Whole Genome Bisulfite Sequencing (WGBS). Genomic DNA was sheared by sonication, end-repaired and then ligated to methylated sequencing adapters. The library fragments were treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymine while leaving methylated cytosines intact. The sequenced fragments were aligned to a bisulfite-converted reference genome. For each assayed cytosine, the number of sequencing reads covering that C and the percentage of those reads that were methylated were reported.

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:
DNA methylation at cytosines across the genome was assayed with Whole Genome Bisulfite Sequencing (WGBS). WGBS was performed on cell lines grown by ENCODE production groups. WGBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology.
Isolation of Genomic DNA: Genomic DNA was isolated from each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation were determined using fluorescent DNA-binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 2 µg of genomic DNA is used to make WGBS libraries.
WGBS Library Construction and Sequencing: WGBS library construction started with sonication of genomic DNA on a Covaris S2 instrument. Sheared ends were then repaired and blunted with DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase in the presence of dATP, dGTP and dTTP. After end repair, Klenow exo- DNA Polymerase was used to add an adenosine as a 3' overhang. Next, a methylated version of the Illumina paired-end adapters was ligated onto the DNA. Adapter-ligated 400 bp genomic DNA fragments were selected using a 2% agarose SizeSelect E-gel. The selected adapter-ligated fragments were treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite-treated DNA was amplified in a final PCR reaction which was optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction, uracils were copied as thymines, resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. These libraries were then sequenced with an Illumina HiSeq 2000 according to the manufacturer's recommendations as paired-end 50 bp reads. Libraries were sequenced to a depth of 600 million aligned reads.
Data Analysis: To analyze the sequence data, Bismark (Krueger and Andrews, 2011) was used to align sequences reads. Generally, each read went through a conversion of Cs to Ts and was then aligned to fully converted plus and minus strands of the hg19 build of the human genome. A few custom refinements were made to the Bismark program. Since these libraries were made in a directional orientation with the first read always being C-poor, we skipped unnecessary alignments to impossible orientations. We also implemented a more stringent uniqueness filter, only allowing reads that have one acceptable alignment (based on default Bowtie parameters) across both strands. Once reads were aligned, the percent methylation was calculated for each cytosine using the original sequence reads. The percent methylation and number of reads is reported for each CpG in the wgEncodeHaibMethylWgbsXXXXCpg.bigBed file and for each non CpG cytosine in the wgEncodeHaibMethylWgbsXXXXNoncpg.bigBed file.

***Previously submitted data files were in error and have been removed. New data files will be added soon.***

first: Richard

last: Myers

first: Florencia

last: Pauli


title: Transcriptional Regulation of the GPX1 Gene by TFAP2C and Aberrant CpG Methylation in Human Breast Cancer

city: St Louis

first: Tiandao

last: Li

state: MO

address: 4444 Forest Park Ave

institute: Washington University

summary: The complexity of gene regulation has created obstacles to defining mechanisms that establish the patterns of gene expression characteristic of the different clinical phenotypes of breast cancer. Transcription factor TFAP2C plays a critical role in the regulation of both estrogen receptor-alpha (ERα) and c-ErbB2/HER2 (Her2). Herein, we performed chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in four breast cancer cell lines representing different clinical phenotypes. Comparing the genomic binding sites for TFAP2C in the various cell lines, we identified that glutathione peroxidase (GPX1) is regulated by TFAP2C through an AP-2 regulatory region in the promoter of the GPX1 gene. Knock-down of TFAP2C, but not the related factor TFAP2A, resulted in an abrogation of GPX1 expression. Selenium-dependent GPX activity correlated with endogenous GPX1 expression, and overexpression of exogenous GPX1 induced GPX activity and significantly increased resistance to tert-butyl hydroperoxide. Methylation of the CpG island encompassing the AP-2 regulatory region was identified in cell lines where TFAP2C failed to bind the GPX1 promoter and GPX1 expression was unresponsive to TFAP2C. Furthermore, in cell lines where GPX1 promoter methylation was associated with gene silencing, treatment with 5-aza-dC (an inhibitor of DNA methylation) resulted in activation of GPX1 RNA and protein expression. Methylation of the GPX1 promoter was identified in approximately 20% of primary breast cancers and a highly significant correlation between TFAP2C and GPX1 expression was confirmed when considering only those tumors with an unmethylated promoter, whereas the related factor, TFAP2A, failed to demonstrate a correlation. The results demonstrate that TFAP2C regulates the expression of GPX1, which influences the redox state and sensitivity to oxidative stress induced by peroxides. Given the established role of GPX1 in breast cancer, the results provide an important mechanism for TFAP2C to further influence oncogenesis and progression of breast carcinoma cells.

overall_design: 4 ChIP-Seq data for TFAP2C in human breast carcinoma cell lines MCF-7, BT-474, MDA-MB-453 and SKBR-3.

first: Mikhail

middle: V

last: Kulak

first: Anthony

middle: R

last: Cyr

first: George

last: Woodfield

last: Bogachek

first: David

middle: H

last: Price

first: Frederick

middle: E

last: Domann

first: Ronald

last: Weigel


title: Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in Uterine Fibroids and Breast Cancer (ChIP-Seq)

status: Public on Sep 11 2012

first: Damian

last: Roqueiro

address: Mattenstrasse 26

department: Biosystems Science and Engineering

institute: ETH Zurich

summary: Analysis of genes regulated by RU486 (an progesterone antagonist) in human breast cancer T47D cells and human uterine leiomyoma smooth muscle cells. The hypothesis is that RU486 inhibits tumor growth by inactivating the transcription of multiple genes which trigger critical signaling pathways to induce tumorigenesis in both breast caner and uterine leomyoma.  Tissue-specific and common patterns of gene regulation may determine the therapeutic effects of antiprogestins in uterine leiomyoma and breast cancer.

overall_design: We applied ChIP-seq to identify PR-interaction sites in T47D breast cancer cells and primary uterine leiomyoma cells treated with RU486.

first: Bulun

last: Serdar

first: Frasor

last: Jonna


title: Genome-wide maps of Six2 and b-catenin in mesenchymal nephron progenitors

city: Cincinnati

first: Joo-Seop

last: Park

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: Self-renewing undifferentiated nephron progenitors express Six2, a transcription factor that is required for their maintenance as undifferentiated progenitors. Differentiation of nephron progenitors is triggered by Wnt/b-catenin signaling. In order to understand how Six2 and Wnt signaling counteract each other, we performed ChIP-seq of Six2 and b-catenin in mesenchymal nephron progenitor cells.

overall_design: Nephron progenitors were FACS-isolated from BAC transgenic Six2GFPcre-positive embryonic kidneys at E16.5. For Six2 ChIP, freshly FACS isolated Six2+ cells were used. For b-catenin ChIP, FACS isolated Six2+ cells were aggregated by centrifugation at 850g for 5min and incubated in 10%FBS/DMEM containing 4uM BIO for 24hrs.

first: Wenxiu

last: Ma

first: Wing

last: Wong

first: Andrew

last: McMahon


title: Interpopulation differences in gene regulation in response to acute thermal stress

city: La Jolla

first: Felipe

last: Barreto

state: California

address: 8750 Biological Grade

department: Marine Biology Research Division

institute: Scripps Institution of Oceanography

summary:
Background:  Geographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance.
Results: Observed differences in gene expression between the southern (San Diego) and the northern (Santa Cruz) populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of up-regulation of genes producing heat shock proteins (Hsps) and genes involved in ubiquitination and proteolysis. Cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were downregulated in both populations. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes.
Conclusions: Marked changes in gene expression were observed in response to acute sublethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations (e.g., cuticle gene regulation), the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s) involved in acute temperature stress may offer at least a partial explanation of latitudinal trends in thermal tolerance observed in Tigriopus.

overall_design: For each population, ~600 copepods were split into two equal samples, one for control and one for treatment.  Each sample was placed in a 50 mL Falcon tube containing 30 mL filtered seawater.  After equilibrating samples to 20 degrees C, each tube was immersed in water bath at its target temperature (control: 20 C; treatment: 35 C) for one hour, and then immersed at 20 C for one hour for recovery.  Copepods were then collected in a net mesh and quickly transferred to a tube containing 5 mL Tri-reagent for standard RNA extraction.

first: Sean

middle: D

last: Schoville

middle: S

first: Gary

last: Moy

first: Anastasia

last: Wolff

last: Burton


title: Alkbh1/Tzfp Repress a Non-Repeat piRNA Cluster in Pachytene Spermatocytes

city: Oslo

first: Jonas

last: Paulsen

state: Foreign

address: Montebello

department: Radium Hospital

country: Norway

institute: Oslo University Hospital

summary: Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also necessary for spermatogenesis in adult testes, however, their function here remains a mystery. Here, we use germ cell isolations and small RNA sequencing to show that most piRNAs in meiotic spermatocytes originate from clusters in intergenic non-repeat regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through mostly unknown processes. We present evidence for a regulatory mechanism for one such cluster, named cluster 1082B, located on chromosome 7 in the mouse genome, containing 788 unique piRNAs. The precursor transcript and individual piRNAs within the cluster are repressed by the Alkbh1 dioxygenase and the transcription repressor Tzfp, which are believed to be interaction partners in testis. We observe more than a thousand-fold upregulation of individual piRNAs in pachytene spermatocytes isolated from Alkbh1-/- and TzfpGTi/GTi testes. Repression is further supported by the identification of a 10 bp Tzfp recognition sequence contained within the precursor transcript. Downregulation of long interspersed elements 1 (LINE1) and intracisternal A-particle (IAP) transcripts in the Alkbh1-/- and TzfpGTi/GTi testes leads us to propose a potential role for the 1082B-encoded piRNAs in transposon silencing.

overall_design: Characterization of small RNAs in mouse pachytene spermatocytes for wild-type (WT) and Alkbh1-/- and TzfpGTi/GTi, and mRNA in mouse pachytene spermatocytes for wild-type (WT) and Alkbh1-/-

first: Line

middle: M

last: Nordstrand

first: Kari

last: Furu

first: Torbjørn

last: Rognes

first: Arne

last: Klungland


title: A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals (RNA-Seq)

city: Worcester

first: Manuel

last: Garber

state: Massachusetts

address: 55 Lake Ave North Worcester

department: Bioinformatics and Integrative Biology

institute: University of Massachusetts Medical School

summary: Dynamic binding of transcription factors to DNA elements specifies gene expression and cell fate, in both normal physiology and disease. To date, our understanding of mammalian gene regulation has been hampered by the difficulty of directly measuring in vivo binding of large numbers of transcription factors to DNA. Here, we develop a high-throughput indexed Chromatin ImmunoPrecipitation (iChIP) method coupled to massively parallel sequencing to systematically map protein-DNA interactions. We apply iChIP to reconstruct the physical regulatory landscape of a mammalian cell, by building genome-wide binding maps for 29 transcription factors (TFs) and chromatin marks at four time points following stimulation of primary dendritic cells (DCs) with pathogen components. Using over 180,000 TF-DNA interactions in these maps, we derive an initial dynamic physical model of a mammalian cell regulatory network. Our data demonstrates that transcription factors vary substantially in their binding dynamics, genomic localization, number of binding events, and degree of interaction with other factors. Further, many of the TF-DNA interactions at stimulus-activated genes are established during differentiation and maintained in a poised state. Functionally, the TFs are organized in a hierarchy of different types: Cell differentiation factors bind most of the genes and remain largely unchanged during the stimulation. A second set of TFs bind already in the un-stimulated and preferentially target induced genes. A third set consists of TF that bind mainly after the stimuli and target specific gene functions. Together these factors determine the magnitude and timing of stimulus induced gene expression. Our method, which allowed us to map routinely temporal binding profiles of dozens of TFs, provides a foundation for future understanding of the mammalian regulatory code.

overall_design: A study of dynamic binding of transcription factors in an immune cell following pathogen stimulation

first: Nir

last: Yosef

first: Raktima

last: Raychowdhury

first: Anne

last: Thielke

first: Joshua

last: Levin

first: Bradley

last: Bernstein

first: Chad

last: Nusbaum

last: Hacohen

first: Aviv

last: Regev

first: Ido

last: Amit


title: A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals


title: LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance

status: Public on Sep 10 2012

first: Gene

last: Yeo

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD


title: A gene expression signature of PDGF-stimulated fibroblasts

city: Majadahonda

first: Cristina

last: Peña

state: Madrid

address: Joaquín Rodrigo,2

country: Spain

institute: Hospital Universitario Puerta de Hierro, Majadahonda

summary: Analysis of platelet-derived growth factor (PDGF)-stimulated fibroblasts. Results provide insight into novel pro-tumorigenic factors induced by PDGF-activation of fibroblasts.

overall_design: 1.2 x 10e6 BJ-hTERT fibroblasts were cultured in 10cm dish with Dulbecco's Modified Eagle Medium (DMEM; Gibco Life Technologies, Gergy-Pontoise, France) containing 1% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 ng/ml) at 37ºC in a 5% CO2 humidified atmosphere during 24h. Cultures were stimulated with or without 20ng/mL PDGF-BB (Peprotech, New Jersey, USA) for another 24h prior to harvest. Unstimulated fibroblasts were used as control samples. Cultures of both unstimulated and stimulated fibroblasts were done in triplicate.


title: LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance (HTS)

summary: LIN28 is a conserved RNA binding protein implicated in pluripotency, reprogramming and oncogenesis. Previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28 binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

overall_design: CLIP-seq for LIN28-V5 in stable human Flp-In-293 cells, and LIN28 in hES cells; strand-specific mRNA-seq for uninfected, control KD, and LIN28 KD human H9 ES cells; and strand-specific smallRNA-seq for uninfected, control KD, and LIN28 KD human H9 ES cells.

first: Stephanie

last: Huelga

first: Melissa

middle: L

last: Wilbert


title: Identification of Cux2 binding sites in female and male mouse liver

city: Boston

first: David

middle: J.

last: Waxman

state: MA

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

summary: Growth hormone (GH)-regulated transcription factors, notably STAT5 and BCL6, play a major role in regulating genes showing sex differences in expression in mouse liver, primarily through their effects in male liver, where male-biased genes are up regulated and many female-biased genes are actively repressed. Here we investigate whether complementary mechanisms, involving up regulation of female-biased genes and down regulation of male-biased genes occur in female liver. To address this question, we identified genes regulated by Cux2, a highly female-specific transcription factor that is repressed in male liver and is induced by the female plasma GH pattern in female liver.  ChIP-seq analysis identified Cux2 binding sites in female liver only, where they are enriched at sites of male-biased DNase hypersensitivity and at genomic regions showing male-enriched STAT5 binding.  Cux2 binding sites were enriched at genes repressed by adenoviral-Cux2, but not at genes induced by adenoviral-Cux2 (see GSE35897), indicating a direct binding mechanism in Cux2 repression but not in Cux2-dependent gene induction. (Published in: TL Conforto et al 2012, Mol Cell Biol. 2012, 32:4611-4627. PubMed PMID: 22966202; PMCID: PMC3486175)

overall_design: Mouse livers were excised from individual male and female. Sonicated, cross-linked liver nuclear chromatin was then used to identify Cux2 binding sites by ChIP-Seq using antibody specific to Cux2, amino acids 356 to 415

first: Tara

last: Conforto

first: Yijing

last: Zhang


title: P6 Mouse Sertoli Cell Small RNA seq

status: Public on Sep 08 2012

city: Reno

first: Nicole

last: Ortogero

state: Nevada

address: 1664 N. Virginia St

department: Physiology and Cell Biology

institute: University of Nevada, Reno

summary: This study annotates the NGS reads in a Sertoli cell sncRNA library.  In addition to known sncRNAs, we also identified numerous novel sncRNAs expressed by Sertoli cells.  Our data suggest that the Sertoli cell sncRNA transcriptome predominantly consists of miRNAs, piRNA-like RNAs, tRNAs and snoRNAs.  This study reports the first comprehensive annotation of the Sertoli cell sncRNA transcriptome.

overall_design: Sertoli cells were purified from post-natal day 6 C57Bl/6 mice using the STAPUT unit gravity sedimentation apparatus.  Small RNA was isolated from cells and a cDNA library was generated for 454 sequencing.  Sequences from FastA files were initially mapped to known sequences and the genome and subsequently annotated upon novel small RNA categorization.

first: Grant

last: Hennig

last: Langille

first: Seungil

last: Ro

first: John

last: McCarrey


title: Argonaute proteins couple chromatin silencing to alternative splicing (RNA IP-Seq)

city: Paris

first: Eric

last: Batsche

address: 7-9, Quai Saint Bernard

department: B2A Biological Adaptation and Ageing - UMR8256 CNRS

country: France

institute: IBPS - Institut Biologie Paris Seine

summary: While Argonaute (AGO) proteins play a major role in transcriptional gene silencing (TGS) in many organisms, their role in the nucleus of somatic mammalian cells remains elusive. Here, we have purified AGO1 and AGO2 chromatin-embedded complexes, and found these proteins associated with previously described partners, but also with chromatin modifiers and, rather unexpectedly, with different splicing factors. Using the CD44 gene as a model for alternative splicing, we show that both AGO1 and AGO2 are required for Protein Kinase C (PKC)-dependent variant exon inclusion. AGO proteins facilitate the spliceosome recruitment and modulate the elongation rate of RNA polymerase II (RNAPII). The recruitment of AGO proteins to CD44 transcribed region is dependent on both the endonuclease Dicer and the chromodomain-containing protein HP1g, and results in locally increased levels of histone H3 lysine 9 (H3K9) methylation on variant exons. Genome wide analysis of splicing in either AGO2 or Dicer null cells showed that the two proteins have similar effects on many splicing events. Finally, sRNAs associated with nuclear AGO2 are mostly in sense orientation relative to protein-coding genes, supporting a role for intragenic antisense non-coding RNAs in the recruitment AGO and splicing factors. Together, our data demonstrate for the first time that the endogenous RNAi pathway is involved in alternative splicing decisions, unravelling a new model in which AGO proteins couple RNAPII elongation and chromatin modification.

overall_design: Deep sequencing of small RNAs (approx. 15-80 nucleotides) bound to either cytoplasmic or chromatin-associated AGO2 complex.

first: M

last: Ameyar-Zazoua

first: C

last: Rachez

last: Souidi

first: R

last: Young

first: N

last: Morozova

first: J

last: Mathieu

last: Descostes

first: JC

last: Andrau

last: Muchardt

first: A

last: Harel-Bellan

first: E

last: Batsché


title: CTCF binding patterns in mouse trophoblast stem cells

city: Chapel Hill

middle: Mauro

last: Calabrese

state: NC

address: 120 Mason Farm Road

department: Pharmacology

institute: UNC-Chapel Hill

summary: This Series reports data from a CTCF ChIP-Seq experiment performed in F1-hybrid mouse trophoblast stem cells (TSCs). The data are part of a larger study examining inactive X gene expression and chromatin states, reported as GEO Series GSE39406. Included for this dataset are FASTQ files, BED alignments and WIG files with coordinates relative to UCSC genome build mm9, and _snp files that report the location of all SNP-overlapping reads

overall_design: Single CTCF ChIP-Seq experiment


title: DNaseI/FAIRE/ChIP Synthesis from ENCODE/OpenChrom(Duke/UNC/UTA)

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
These tracks display a synthesis of evidence from different assays as part of the four Open Chromatin track sets. This track displays open chromatin regions and/or transcription factor binding sites identified in multiple cell types by one or more complementary methodologies, DNaseI hypersensitivity (HS) (Duke DNaseI HS), Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) (UNC FAIRE), and chromatin immunoprecipitation (ChIP) for select regulatory factors (UTA TFBS). Each methodology was performed on the same cell type using identical growth conditions. (Note: Data for some or all ChIP experiments may not be available for all cell types). Regions that overlap between methodologies identify regulatory elements that are cross-validated indicating high confidence regions.  In addition, multiple lines of evidence suggest that regions detected by a single assay (e.g., DNase-only or FAIRE-only) are also biologically relevant (Song et al., submitted).
DNaseI HS data: DNaseI is an enzyme that has long been used to map general chromatin accessibility, and DNaseI "hypersensitivity" is a feature of active cis-regulatory sequences.The use of this method has led to the discovery of functional regulatory elements that include promoters, enhancers, silencers, insulators, locus control regions, and novel elements. DNaseI hypersensitivity signifies chromatin accessibility following binding of trans-acting factors in place of a canonical nucleosome.
FAIRE data: FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements) is a method to isolate and identify nucleosome-depleted regions of the genome. FAIRE was initially discovered in yeast and subsequently shown to identify active regulatory elements in human cells (Giresi et al., 2007). Similar to DNaseI HS, FAIRE appears to identify functional regulatory elements that include promoters, enhancers, silencers, insulators, locus control regions and novel elements.
ChIP data: ChIP (Chromatin Immunoprecipitation) is a method to identify the specific location of proteins that are directly or indirectly bound to genomic DNA. By identifying the binding location of sequence-specific transcription factors, general transcription machinery components, and chromatin factors, ChIP can help in the functional annotation of the open chromatin regions identified by DNaseI HS mapping and FAIRE.
Input data: As a background control experiment, we sequenced the input genomic DNA sample that was used for ChIP. Crosslinked chromatin is sheared and the crosslinks are reversed without carrying out the immunoprecipitation step. This sample is otherwise processed in a manner identical to the ChIP sample as described below. The input track is useful in revealing potential artifacts arising from the sequence alignment process such as copy number differences between the reference genome and the sequenced samples, as well as regions of poor sequence alignability.

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:
For each site, the maximum F-Seq Density Signal value has been calculated for each assay that was performed in that cell type. F-Seq employs Parzen kernel density estimation to create base pair scores (Boyle et al., 2008b). Significant regions, or peaks, were determined by fitting the data to a gamma distribution to calculate p-values. Contiguous regions where p-values were below a 0.05 (DNaseI HS, ChIP) or 0.1 (FAIRE) threshold were considered significant. See assay specific description pages ( Duke DNaseI HS, UNC FAIRE and UTA TFBS) for more details.
A Fisher's Combined P-value for DNaseI HS and FAIRE was calculated using Fisher's combined probability test. First, a test statistic is calculated using the formula x^2 = -2*sum(ln(pi)) where pi are the p-values calculated for DNaseI HS and FAIRE. X2 follows a chi-squared distribution, thus a combined p-value can be assigned to this test statistic.

first: Terry

last: Furey

first: Zhancheng

last: Zhang

first: Lingyun

last: Song

first: Greg

last: Crawford

first: Paul

last: Giresi

first: Jason

last: Lieb

first: Zheng

last: Liu

first: Ryan

last: McDaniell

first: Bum-Kyu

last: Lee

first: Vishy

last: Iyer

last: Flicek

first: Damian

last: Keefe

first: Ewan

last: Birney

first: Stefan

last: Graf


title: RNA-Seq of control and ELL2-depleted MPC11 plasmacytoma cell lines

status: Public on Sep 07 2012

city: New York

first: Tarmo

last: Äijö

address: 162 5th Avenue

department: Center for Computational Biology

institute: Flatiron Institute

summary: We report RNA-Seq data from MPC11 plasmacytoma cell lines transduced with either control hairpins (shGFP) or hairpins targeting the transcriptional elongation factor ELL2.  ELL2 has been identified as regulating the alternative splicing and polyadenylation of immunoglobulin pre-mRNA, and is highly expressed by plasma cells but not other B cells.  Notably, ELL2 drives the accumulation of transcripts encoding secreted immunoglobulin at the expense of membrane-associated transcripts.  This study sought to examine the impact of ELL2 on mRNA processing at the transcriptome level, and found that ELL2 controls the alternative processing of 12% of annotated transcripts in MPC11 cells.

overall_design: Examination of pre-mRNA  splicing patterns in either control or ELL2-depleted cells

first: Micah

last: Benson

first: Anjana

last: Rao


title: A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals (ChIP-Seq)

first: Manuel

last: Garber

first: Nir

last: Yosef

first: Raktima

last: Raychowdhury

first: Anne

last: Thielke

first: Bradley

last: Bernstein

first: Chad

last: Nusbaum

last: Hacohen

first: Aviv

last: Regev

first: Ido

last: Amit


title: Corepressors (NCoR and SMRT) as well as Coactivators are Recruited to Positively Regulated 1α,25-Dihydroxyvitamin D3-Responsive Genes

status: Public on Sep 05 2012

city: Madison

first: Mark

middle: B

last: Meyer

state: WI

address: 1415 Linden Dr.

department: Nutritional Sciences

institute: University of Wisconsin-Madison

summary: Transcription factors require coactivators and corepressors to modulate transcription in mammalian cells.  The vitamin D receptor (VDR) utilizes coactivators and corepressors to gain tight control over the activity of a diverse set of genes that can regulate calcium transport, slow proliferation and promote immune responses.  We have recently established the VDR/RXR cistrome in human colon cancer cells and have linked these binding sites to the genes that are regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3).  In additional studies described herein, we demonstrate that the coactivators SRC1, CBP and MED1 are recruited to upregulated genes to facilitate transcription as expected.  SRC1 was the most highly correlated to VDR/RXR binding (50%).  However, we also found that corepressor molecules such as NCoR and SMRT were present along with SRC1, CBP or MED1 at these 1,25(OH)2D3 activated gene enhancers.  Interestingly, genome-wide NCoR binding mimicked VDR binding by increasing its association with VDR binding in response to 1,25(OH)2D3 treatment.  Overall, these data indicate a complex role for corepressor and coactivator complexes in the activation or active repression of 1,25(OH)2D3 responsive genes.

overall_design: 5 coregulators are analyzed by ChIP-seq. The Input from GSE31939 was used as the normalizing factor for all transcription factor IPs.  If lanes had more than 1 replicate, these lanes were combined for greater read depth.

first: J

middle: W

last: Pike


title: Genome-wide Profiling of Progesterone Receptor and GATA2 Binding in the Mouse Uterus

status: Public on Sep 06 2012

city: Research Triangle Park

first: Franco

last: DeMayo

address: Building 101, Room F191 111 T.W. Alexander Drive

department: Reproductive and Developmental Biology Laboratory

institute: National Institute of Environmental Health Sciences

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

overall_design: Refer to individual Series


title: Genome-wide Profiling of Progesterone Receptor and GATA2 Binding in the Mouse Uterus [ChIP-Seq]

summary: Progesterone (P4) signaling through its nuclear transcription factor, the progesterone receptor (PR), is essential for normal uterine function.  Although deregulation of PR mediated signaling is known to underscore uterine dysfunction and a number of endometrial pathologies, the early molecular mechanisms of this deregulation are unclear. To address this issue, we have defined the genome-wide PR and GATA2 cistrome in the murine uterus using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). In uteri of ovariectomized mice, we identified 6367 PR binding sites in the absence of P4 ligand; however, this number increased at nearly three fold (18,432) following acute P4 exposure. Sequence analysis revealed that approximately 73% of these binding sites contain a progesterone response element (PRE) or a half-site motif recognized by the PR. Many previously identified P4 target genes known to regulate uterine function were found to contain PR binding sites, confirming the validity of our methodology.  In addition we identified 46,183 GATA2 binding sites in P4 treatment conditions with 7,954 binding sites overlapping that of the PR.

overall_design: Examination of PR and Gata2 binding in whole or epithelial isolated mouse uterine tissue upon acute vehicle/P4 treatement

first: Cory

middle: A

last: Rubel

first: Rainer

last: Lanz

first: Ramakrishna

last: Kommagani

first: Heather

last: Franco

first: Sally

last: Camper

first: Ertug

last: Kovanci

first: Qiang

last: Tong

first: Jae

last: Jeong

first: John

last: Lydon

first: Francesco

middle: J


title: Robust 4C-seq data analysis to screen for regulatory DNA interactions

status: Public on Sep 04 2012

city: Rehovot

first: Gilad

last: Landan

address: 234 Herzl Street

country: Israel

institute: Weizmann Institute of Science

summary: Regulatory DNA elements can control expression of distant genes via physical interactions. Here, we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using Chromosome Conformation Capture combined with high-throughput sequencing (4C-seq) data. Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation.

overall_design: A high resolution 4C-seq protocol involving two restriction digests and a revised analysis pipeline was applied to several viewpoints in four genomic loci (the well-characterized alpha-globin and beta-globin loci, and the novel Oct4 and Satb1 loci), allowing robust detection of physical interactions between regulatory DNA elements.

first: Harmen

last: van de Werken


title: Unraveling the KNOTTED1 regulatory network in maize meristems (ChIP-seq)

city: East Lansing

first: Erich

last: Grotewold

address: 603 Wilson Road

institute: Michigan State University

summary: KNOTTED1(KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. Despite their importance for plant development, the genomic network targeted by KNOX proteins is poorly understood. Using ChIP-seq, we defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci. To understand how these binding occupancies correlate with changes in transcriptional regulation, we performed RNA-seq on immature ears and tassels, and compared expression profiles between normal and loss-of-function kn1 plants, in addition to immature leaves from normal and gain-of-function Kn1 plants. We found that 643 of the KN1 targets were modulated in one or multiple tissues, with a strong enrichment for transcription factors (including other homeobox genes) and genes participating in several hormonal pathways, most significantly auxin, implicating KN1 at the crossroads of plant hormone signaling. The loss-of-function kn1 phenotype is reminiscent of auxin mutants and kn1 mis-expression in leaves correlates with increased auxin signaling. Our results demonstrate that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function.

overall_design: ChIP-seq was performed using ear primordia and tassel primordia. Input DNA from each sample was used as a normalization control

first: Nathalie

last: Bolduc

first: Alper

last: Yilmaz

first: Maria

middle: K

last: Mejia-Guerra

first: Kengo

last: Morohashi

first: Devin

last: O’Connor

first: Sarah

last: Hake


title: A role for Fkbp6 and the chaperone machinery in piRNA amplification and transposon silencing

city: Gneveva

first: Ramesh

last: Pillai

address: 30, Quai Ernest-Ansermet

department: Department of Molecular Biology

institute: University of Geneva

summary: Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other co-chaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be by-products of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.

sample_organism:
Bombyx mori
Mus musculus

platform_organism:
Bombyx mori
Mus musculus

overall_design: Small RNAs were purified for preparation of high-throughput sequencing libraries. All libraries except JX56, JX57, JX36 and JX37 were generated from isolated small RNAs obtained by immunoprecipitation of the indicated proteins (Mili, Miwi2, Siwi or Ago3). JX56 and JX57 were prepared from small RNAs extracted from mouse testis. JX36 and JX37 were prepared from polyA+ RNAs extracted from the Bombyx mori cell line BmN4. Libraries prepared from mouse were prepared from mice having the indicated genotype. BmN4 cells were treated with the Hsp90 inhibitor geldanamycin (GA) before performing the immunoprecipitation in the indicated libraries. This lead to accumulation to a short species of small RNAs (16nt long) in Ago3 complexes which we called ping-pong by-product. Details can be found in Xiol et al. 2012, Molecular Cell.

first: Jordi

last: Xiol

first: Elisa

last: Cora

first: Rubina

last: Koglgruber

first: Shinichiro

last: Chuma

first: Sailakshmi

last: Subramanian

first: Mihoko

last: Hosakawa

first: Michael

last: Reuter

first: Zhaolin

last: Yang

first: Philipp

last: Berninger

first: Andres

last: Palencia

first: Vladimir

last: Benes

first: Josef

last: Penninger

first: Ravi

last: Sachidanandam


title: New partners in regulation of gene expression: the Enhancer of Trithorax and Polycomb Corto interacts with methylated Ribosomal Protein L12 via its chromodomain.

first: Stéphane

last: LE CROM

address: 46 rue d'Ulm

department: Biology Institute - IBENS

institute: École normale supérieure

summary: Chromodomains are found in many regulators of chromatin structure. Most of them recognize methylated histones. Here, we investigate the role of the Corto chromodomain. This Drosophila melanogaster Enhancer of Polycomb and Trithorax is involved in both silencing and activation of gene expression. Overexpression of Corto chromodomain (CortoCD) in transgenic flies show that this domain is critical for Corto function and behaves as a chromatin-targeting module. Mass spectrometry analysis of peptides pulled down by CortoCD from nuclear extracts reveals that they correspond to nuclear ribosomal proteins (RPs). Notably, CortoCD binds with high affinity RPL12 tri-methylated on lysine 3 (RPL12K3me3) as demonstrated by real-time interaction analyses. Co-localization of Corto and RPL12 with active epigenetic marks on polytene chromosomes suggests that they are involved in fine-tuning transcription of genes located in open chromatin. Hence, pseudo-ribosomal complexes composed of various RPs might participate in regulation of gene expression in connection with chromatin regulators. RNA-seq analysis of wing imaginal discs overexpressing either Corto or RPL12 show that most deregulated genes are shared by both factors. Interestingly, these common targets are enriched in RP genes suggesting that Corto and RPL12 are involved in dynamic coordination of ribosome biogenesis.

overall_design: To address the role of Corto and RPL12 in regulation of transcription, we deep-sequenced transcripts of wing imaginal discs from third instar larvae over-expressing either FH-cortoCD or RpL12-Myc under control of the wing-specific scalloped::Gal4 driver (sd::Gal4>UAS::FH-cortoCD or sd::Gal4>UAS::RpL12-Myc). Total RNA from FH-cortoCD or RpL12-Myc, the sd::Gal4/+ control or a w1118 reference line were isolated from pools of wing imaginal discs and subjected to RNA-seq on an Illumina high throughput sequencer.

first: Anne

last: Coléno-Costes

first: Suk

last: Min Jang

first: Augustin

last: de Vanssay

first: Julien

last: Rougeot

first: Tahar

last: Bouceba

first: Neel

last: Randsholt

first: Jean-Michel

last: Gibert

last: Le Crom

first: Emmanuèle

last: Mouchel-Vielh

first: Sébastien

last: Bloyer

first: Frédérique

last: Peronnet


title: Adiponectin regulates expression of hepatic genes critical for glucose and lipid metabolism.

city: Cambridge

first: Qingqing

last: Liu

address: 9 Cambridge Center

institute: Whitehead Institute

summary: The effects of adiponectin on hepatic glucose and lipid metabolism at transcriptional level are largely unknown. We profiled hepatic gene expression in adiponectin knockout (KO) and wild-type (WT) mice by RNA-Seq. Comparing to WT mice, adiponectin KO mice exhibited decreased mRNA expression of rate-limiting enzymes in several important glucose and lipid metabolic pathways including glycolysis, TCA cycle, fatty-acid activation and synthesis, triglyceride synthesis and cholesterol synthesis. In addition, binding of the transcription factor Hnf4a to DNAs encoding several key metabolic enzymes was reduced in KO mice, suggesting that adiponectin might regulate hepatic gene expression via Hnf4a.  Phenotypically, adiponectin KO mice possessed smaller epididymal fat pads and showed reduced body weights comparing to WT mice. When fed a high fat diet, adiponectin KO mice showed significantly reduced lipid accumulation in the livers. These lipogenic defects are consistent with the downregulation of lipogenic genes in the KO mice.

overall_design: Mice were fasted overnight before euthanization. Liver tissues from WT or adiponectin KO male mice (n = 9-10) at 12 weeks of age were harvested and subjected to total RNA extraction using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA).  Total RNA from nine to ten mice of the same strain was pooled together as one biological sample. The mRNA sequencing samples were prepared using the Illumina sample preparation protocol (RS-930-1001, Illumina, Inc. San Diego, CA). The cDNA fragments of 200–250 bp were purified on an agarose gel and then enriched by PCR with Phusion polymerase. The cDNA libraries were sequenced by an Illumina Genome analyzer II at the Whitehead Genome Technology Core.


title: Genome-wide measurement of RNA folding energies

status: Public on Sep 09 2012

city: Stanford

first: Kun

last: Qu

address: 269 Campus Dr. CCSR 2150

department: Dermatology

institute: Stanford University

summary: RNA structural transitions are important in the function and regulation of RNAs. Here, we reveal a layer of transcriptome organization in the form of RNA folding energies. By probing yeast RNA structures at different temperatures, we obtained relative melting temperatures (Tm) for RNA structures in over 4000 transcripts. Specific signatures of RNA Tm demarcated the polarity of mRNA open reading frames, and highlighted numerous candidate regulatory RNA motifs in 3' untranslated regions. RNA Tm distinguished non-coding versus coding RNAs, identified mRNAs with distinct cellular functions. We identified thousands of putative RNA thermometers, and their presence is predictive of the pattern of RNA decay in vivo during heat shock. The exosome complex recognizes unpaired bases during heat shock to degrade these RNAs, coupling intrinsic structural stabilities to gene regulation. Thus, genome-wide structural dynamics of RNA can parse functional elements of the transcriptome and reveal diverse biological insights.

overall_design: RNA structure probing at 5 different temperatures (23°C , 30°C , 37°C , 55°C and 75°C) using RNase V1 on polyA-selected log phase S288C yeast RNAs was followed by library construction using a modified SOLiD small RNA cloning kit and deep sequencing on the SOLiD platform. We performed 2 biological replicates for each temperature.

first: Yue

last: Wan

first: Zhengqing

last: Ouyang

first: Michael

last: Kertesz

first: Jun

last: Li

first: Robert

last: Tibshirani

first: Debora

last: Makino

middle: C

last: Nutter

first: Eran

last: Segal

first: Howard

middle: Y

last: Chang


title: RNA sequencing revealed novel actors of the acquisition of drug resistance in Candida albicans

city: PARIS

first: sophie

last: lemoine

department: Biologie

institute: Ecole Normale Supérieure

summary: Drug susceptible clinical isolates of Candida albicans frequently become highly tolerant to drugs during chemotherapy, with dreadful consequences on patient health. We used RNA sequencing (RNA-seq) to analyze the transcriptomes of a CDR (Candida Drug Resistance) strain and its isogenic drug sensitive counterpart. RNA-seq unveiled differential expression of 228 genes including a) genes  previously identified as involved in CDR, b) genes not previously associated to the CDR phenotype, and c) novel transcripts whose function as a gene is uncharacterized. In particular, we show for the first time that CDR acquisition is correlated with an overexpression of the transcription factor encoding gene CZF1. CZF1 null mutants were sensitive to many drugs, independently of known multidrug resistance mechanisms. We show that CZF1 acts as a repressor of β-glucan synthesis, thus negatively regulating cell wall integrity. Finally, our RNA-seq data allowed us to identify a new transcribed region, upstream of the TAC1 gene, which encodes the major CDR transcriptional regulator. Our results open new perspectives to the role of Czf1 and to our understanding of the transcriptional and post-transcriptional mechanisms that lead to the acquisition of drug resistance in C. albicans, with potential future improvements of therapeutic strategies.

overall_design: RNA sequencing was performed on 2 Candida albicans strains (Gu4 and Gu5). For each strain, we sequenced 2 biological replicates.

first: Sanjiveeni

last: Dhamgaye

first: Maria

last: Bernard

first: Gaelle

last: Lelandais

first: Odile

last: Sismeiro

first: Sophie

last: Lemoine

first: Jean-Yves

last: Coppée

first: Stéphane

first: Rajendra

last: Prasad

first: Frederic

last: Devaux


title: Novel Foxo1-dependent Transcriptional Programs Control Treg Cell Function [ChIP-Seq]

first: Willey

last: Liao

address: 101 Avenue of the Americas

department: Bioinformatics

institute: New York Genome Center

summary: Regulatory T (Treg) cells characterized by expression of the transcription factor forkhead box P3 (Foxp3) maintain immune homeostasis by suppressing self-destructive immune responses1-4.  Foxp3 operates as a late acting differentiation factor controlling Treg cell homeostasis and function5, whereas the early Treg cell lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors6-10. However, whether Foxo proteins act beyond the Treg cell commitment stage to control Treg cell homeostasis and function remains largely unexplored.  Here we show that Foxo1 is a pivotal regulator of Treg cell function.  Treg cells express high amounts of Foxo1, and display reduced T-cell receptor-induced Akt activation, Foxo1 phosphorylation, and Foxo1 nuclear exclusion.  Mice with Treg cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to Foxp3-deficient mice, but without the loss of Treg cells.  Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the proinflammatory cytokine Ifng, that do not appear to be directly regulated by Foxp3.  These findings demonstrate that the evolutionarily ancient Akt-Foxo1 signaling module controls a novel genetic program indispensable for Treg cell function.

overall_design: Treg cells were isolated from wild-type B6 mice or Foxo1tagBirA mice in which foxo1 is endogenously biotinylated. Foxo1 binding targets in Treg cells were identified by using Foxo1 antibody- and Streptavidin- ChIP-Seq approaches.

first: Weiming

last: Ouyang

first: Will

first: Chong

last: Luo

last: Yin

first: Morgan

last: Huse

first: Myoungjoo

middle: V

last: Kim

first: Min

last: Peng

first: Pamela

last: Chan

first: Qian

last: Ma

first: Yifan

last: Mo

first: Dies

last: Meijer

first: Keji

last: Zhao

first: Alexander

last: Rudensky

first: Gurinder

last: Atwal

middle: Q

last: Zhang

first: Ming

middle: O


title: RNA-Sequencing from ventral mid brain and striatum of paraquat, pyridaben and paraquat+maneb mice models of Parkinson's disease.

first: Seema

last: Gollamudi

state: New York

address: 525 East 68th Street A501

department: Neurology and Neuroscience

institute: Weill Cornell Medical College

summary: We report RNA-sequencing from ventral mid brain and striatum from paraquat, pyridaben and paraquat+maneb mice models of Parkinson's disease. We observed several differentially expressed genes upon pesticide exposure which we analyzed by pathway analysis.

overall_design: We examined 3 replicates each for ventral mid brain and striatum per pesticide for RNA-Seq

first: M

middle: F

last: Beal


title: Human-specific patterns of gene expression in the brain (RNA-Seq)

city: Dallas

first: Genevieve

last: Konopka

state: TX

address: 5323 Harry Hines Blvd.

department: Neuroscience

institute: UT Southwestern Medical Center

summary: We identified human-specific gene expression patterns in the brain by comparing expression with chimpanzee and rhesus macaque.

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens

overall_design: DpnII tag-based libraries were generated from human, chimpanzee, and rhesus macaque brain (frontal pole, hippocampus, caudate nucleus), and sequenced using an Illumina Genome Analyzer.

first: Tara

last: Friedrich

first: Daniel

middle: H

last: Geschwind


title: Novel Foxo1-dependent Transcriptional Programs Control Treg Cell Function

first: Willey

last: Liao


title: A comparative epigenomics approach reveals REST as a mediator of Polycomb reprogramming during neuronal differentiation

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

institute: Friedrich Miescher Institute for Biomedical Research

summary: Transcription factors (TFs) in concert with chromatin pathways stably reset transcriptional programs during differentiation. Yet we know little how local sites of chromatin reprogramming are specified and how the estimated 3000 TF encoded in mammalian genomes contribute to chromatin dynamics. To identify candidate TFs we developed an integrated computational approach (Epi-MARA) that models chromatin dynamics in terms of predicted transcription factor binding sites and show that it correctly predicts key TFs involved in epigenome reorganization.  When applied to a time course of genome-wide H3 lysine 27 trimethylation (H3K27me3), a chromatin mark set by the Polycomb system, during neuronal differentiation of murine stem cells Epi-MARA predicted that the repressive transcription factor REST contributes to a gain of H3K27me3 at a subset of promoters during the transition from the stem to the progenitor state. To test this prediction we identified, genome-wide, the actual binding sites of REST and H3K27me3 during the differentiation in cells that are either wildtype or in which REST had been deleted. REST indeed localizes to a subset of sites that gain H3K27me3 in progenitors. Importantly, absence of REST in trans leads to a loss of H3K27me3 predominantly in the neuronal progenitor state and specifically at those regions where REST was bound. This function further requires REST binding sites in cis as their mutation leads to substantial loss of H3K27me3.   Taken together we provide a novel approach to identify epigenome and TF crosstalk during cellular reprogramming and prove experimentally the prediction that REST acts as an important recruiter of Polycomb repression during early steps of neurogenesis.

overall_design: Dataset comprises of 15 ChIP-seq samples using chromatin from embryonic stem (ES) and neuronal progentor (NP) of wildtype and RESTko cells, which was immunoprecipitated, using antibodies against REST, H3K27me3, or Suz12

first: Anne

last: Schoeler

first: Michael

last: Stadler

first: Erik

last: van Nimwegen

last: Schübeler

first: Christian

last: Beisel


title: Role of REST during neuronal differentiation


title: sRNA profiling during Oxytricha conjugation

status: Public on Sep 01 2012

city: Princeton

first: Wenwen

last: Fang

state: NJ

address: 323 Guyot Hall, Washington Rd

department: EEB

institute: Princeton University

summary:
In Oxytricha, the somatic genome is responsible for vegetative growth, while the germline contributes DNA to the next sexual generation. Somatic nuclear development eliminates all transposons and other so-called "junk DNA", which constitute ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally-deleted regions leads to their retention in subsequent generations. Our findings highlight small RNAs (sRNAs) as powerful transgenerational carriers of epigenetic  information for genome programming.

The backcross study here shows that the mating between an IES+ strain with the wild-type stain produces corresponding IES-containing sRNAs at 19 hr, and we provided the mapping to and the sequences of the specific loci of interest in the submission. As a control, wild-type cells do not produce such IES-containing sRNAs, and this analysis can be pulled out from the GSE35018 study since we provided mapping to the whole genome.

The purpose of the 20 hr total sRNA sequencing study here is to show that the class of 27 nt sRNA is the major species of total sRNAs in Oxytricha at 20 hr, which we sequenced previously from Otiwi1-associated sRNAs at 12, 19, 23, and 30 hr (GSE35018).  In addition, there is a less abundant class of small RNAs of 21-22 nt. These two classes are obvious by simply plotting the length distribution of the sRNA sequences.

overall_design:
We sequenced sRNAs from Contig22226.0 IES1+ strain backcrossed to wild-type parental strain at 19hr post-mixing, and found corresponding IES-containing sRNAs. As a control, wild-type cells do not produce such IES-containing sRNAs (see GSE35018).

Total RNA from the backcrossing at 19hr were isolated with mirVana small RNA extraction kit (Ambion), and directly used for making Illumina sRNA libraries.

Oxytricha total small RNA (sRNA) sequencing at 20 hr post conjugation shows that a class of 27 nt, 5'-U sRNAs dominates the sRNA population at 20 hr, and this class of sRNAs associate with Otiwi1 (see GSE35018 for Otiwi1-interacting sRNAs in Oxytricha).  In addition, a much less abundant class of 21-22 nt sRNAs is present according to the length distribution.

first: W

first: J

last: Bracht


title: RNA-seq quantification of expression changes upon unc-62 knockdown in adult Caenorhabditis elegans.

first: Eric

last: Van Nostrand

address: 279 Campus Drive B341

department: Developmental Biology

summary: We performed RNA-seq to quantify gene expression changes in adult worms upon knockdown of transcription factor unc-62/Homothorax. unc-62 is a developmental regulator that binds proximal to age-regulated transcripts and modulates lifespan. In the intestine (in which tissue-specific unc-62 knockdown increases lifespan), we identify multiple effects of unc-62 knockdown linked to extension of longevity. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity and become toxic in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other.

overall_design: mRNA profiling by Illumina HiSeq of 3 biological replicates of day 4 adult Caenorhabditis elegans that were fed either control or unc-62 RNAi beginning at day 1 of adulthood.

first: Stuart

last: Kim


title: Retinoic Acid-Induced DR2 Alu Transcripts in Stem Cells Generate Functional Small RNAs that Mediate Target mRNA Degradation

city: La Jolla

first: MICHAEL

middle: G

last: ROSENFELD

address: 9500 Gilman Drive

institute: HHMI/UCSD

summary: While liganded nuclear receptors are well established to regulate Pol II protein-coding transcription units, their role in regulation of DNA repeats remains largely unknown. Here, we report that ~2-3% of the ~1-200,000 human DR2 Alu repeats, particularly those in proximity to retinoic acid-activated Pol II transcription units, are bound and activated by retinoic acid receptor in human embryonic stem cells, triggering their Pol  III-dependent transcription. The non-coding DR2 Alu transcripts are processed in a Dicer-dependent fashion into a series of small RNA products with sizes ranging ~28-65nt and exhibit substantial co-localization with P bodies.  These small RNAs cause degradation of a subset of mRNAs critical for the "stem cell" state, which harbor complimentary sequences in their 3' untranslated regions. This regulation requires Ago3- dependent stabilization of full-length and processed DR2 Alu transcripts, and recruitment of Ago3-associated decapping complexes to the target mRNAs. Thus, the RAR/Pol III-dependent DR2 Alu transcriptional program in stem cells serves as a functional complement to the RAR/Pol II-dependent counterpart.

overall_design: ChIP-seq of H3K36me3 samples before and after RA (retinoic acid) treatments.

first: Qidong

last: Hu

first: Michele

last: Trabucchi

first: Bogdan

last: Tanasa

first: Christopher

last: Glass

first: Kenneth

last: Ohgi

first: David

last: Rose

last: Rosenfeld


title: Unraveling the KNOTTED1 regulatory network in maize meristems

first: Erich

last: Grotewold

state: MI

overall_design: For loss-of-function mutant, kn1-e1 homozygote were compared to heterozygote or wild-type siblings (ears, tassels and shoot apical meristem). For gain-of-function mutant, Kn1-N homozygote were compared to wild-type siblings.Kn1-N heterozygote were compared to wild-type siblings. For each genotype and tissue, 2 biological replicates were sequenced, except for tassels (3 biological replicates). The whole data set thus includes a total of 20 samples/libraries.

first: Nathalie

last: Bolduc

first: Alper

last: Yilmaz

first: Maria

last: Mejia-Guerra

first: Kengo

last: Morohashi

first: Devin

last: O’Connor

first: Sarah

last: Hake


title: Multiple insert size paired-end sequencing for deconvolution of complex transcriptomes

status: Public on Aug 31 2012

first: Philipp

last: Drewe

address: 417 East 68th Street

department: Computational Biology

institute: Memorial Sloan Kettering Cancer Center

summary: Deep sequencing of transcriptomes allows quantitative and qualitative analysis of many RNA species in a sample, with parallel comparison of expression levels, splicing variants, natural antisense transcripts, RNA editing and transcriptional start and stop sites the ideal goal. By computational modeling, we show how libraries of multiple insert sizes combined with strand-specific, paired-end (SS-PE) sequencing can increase the information gained on alternative splicing, especially in higher eukaryotes. Despite the benefits of gaining SS-PE data with paired ends of varying distance, the standard Illumina protocol allows only non-strand-specific, paired-end sequencing with a single insert size. Here, we modify the Illumina RNA ligation protocol to allow SS-PE sequencing by using a custom pre-adenylated 3’ adaptor. We generate parallel libraries with differing insert sizes to aid deconvolution of alternative splicing events and to characterize the extent and distribution of natural antisense transcription in C. elegans. Despite stringent requirements for detection of alternative splicing, our data increases the number of intron retention and exon skipping events annotated in the Wormbase genome annotations by 127 % and 121 %, respectively. We show that parallel libraries with a range of insert sizes increase transcriptomic information gained by sequencing and that by current established benchmarks our protocol gives competitive results with respect to library quality.

overall_design: Sequencing of mRNA from C. elegans with libraries of four differing insert sizes

first: Lisa

last: Smith

last: Hartmann

first: Regina

last: Bohnert

first: Andre

last: Kahles

first: Christa

last: Lanz

first: Gunnar

last: Rätsch


title: Metabolic regulation of ‘Ca. Methylacidiphilum fumariolicum’ SolV cells grown under different nitrogen and oxygen limitations

city: Nijmegen

first: Huub

last: Op den Camp

address: Heyendaalseweg 135

department: Microbiology

country: Netherlands

web_link: http:// http://www.ru.nl/microbiology/

institute: Radboud University Nijmegen

summary: Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of ‘Ca. Methylacidiphilum fumariolicum’ strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH4 is much more diverse than has previously been assumed in terms of ecology, phylogeny and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of ‘Ca. M. fumariolicum’ SolV cells growing at μmax in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N2 fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem.

overall_design: Cells grown under 3 different conditions were harvested by centrifugation and 3.1 mg dry weight cells were used for isolation of mRNA, and subsequent synthesis of cDNA (328 ng). The cDNA was used for Illumina sequencing on a Illumina Genome.Analyser II

first: Ahmad

last: Khadem

first: Arjan

last: Pol

first: Adam

middle: S

last: Wieczorek

first: Mike

last: Jetten


title: ENCODE PSU Hardison RnaSeq

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 (Ross Hardison mailto:rch8@psu.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).
Knowledge of the function of genomic DNA sequences comes from three basic approaches. Genetics uses changes in behavior or structure of a cell or organism in response to changes in DNA sequence to infer function of the altered sequence. Biochemical approaches monitor states of histone modification, binding of specific transcription factors, accessibility to DNases and other epigenetic features along genomic DNA. In general, these are associated with gene activity, but the precise relationships remain to be established. The third approach is evolutionary, using comparisons among homologous DNA sequences to find segments that are evolving more slowly or more rapidly than expected given the local rate of neutral change. These are inferred to be under negative or positive selection, respectively, and interpreted as DNA sequences needed for a preserved (negative selection) or adaptive (positive selection) function.
The ENCODE project aims to discover all the DNA sequences associated with various epigenetic features, with the reasonable expectation that these will also be functional (best tested by genetic methods). However, it is not clear how to relate these results with those from evolutionary analyses. The mouse ENCODE project aims to make this connection explicitly and with a moderate breadth. Assays identical to those being used in the ENCODE project are performed in cell types in mouse that are similar or homologous to those studied in the human project. Thus we will be able to discover which epigenetic features are conserved between mouse and human, and we can examine the extent to which these overlap with the DNA sequences under negative selection. The contribution of DNA with a function preserved in mammals versus that with a function in only one species will be discovered.
One of the epigenetic features most closely related to genomic activity is the production of stable RNA, including transcripts from both protein-coding genes and noncoding transcripts. These genomic compilations of transcripts, or transcriptomes, are primary determinants of the way cells function, respond and differentiate, both by the production of proteins translated from coding transcripts and the regulatory activity of untranslated non-coding transcripts. Non-coding RNA's regulate gene expression through diverse mechanisms ranging from reducing chromatin accessibility (affecting large regions or whole chromosomes) to precise fine-tuning of transcription from specific genes, e.g. via RNAi.
Even though a large proportion of mammalian genomes is transcribed, many of the transcribed segments have yet to be assigned any function. The ENCODE project aims to create a comprehensive, quantitative annotation of the human transcriptome in several cell and tissue types as well as to understand regulation of transcriptomes by establishing the relationship between regulatory factors and their targets. Mapping the mouse transcriptome in similar tissues will allow us to discern conservation of transcriptome profiles between mouse and human and to discover species-specific transcription patterns, and to infer conserved versus species-specific regulatory mechanisms. The results will have a significant impact on our understanding of the evolution of gene regulation.

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 (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell/mouse).
Total RNA was extracted from 5-10 million cells using TRIzol reagent. This was followed by mRNA selection, fragmentation and cDNA synthesis, which were performed as described previously (Mortazavi et al., 2009). Double-stranded cDNA samples were processed for library construction for Illumina sequencing, using the Illumina ChIP-seq Sample Preparation Kit.
Strand-specific libraries were generated in a similar manner, except for a couple of modifications described previously (Parkhomchuk et al., 2009). Briefly, instead of dTTP, dUTP was used during second-strand cDNA synthesis to label the second-strand cDNA. During library preparation, the dUTP-labeled cDNA was treated with Uracil N Glycosylase, prior to the PCR amplification step. This was done to remove uracil from the second-strand, following which the DNA was subjected to high heat to facilitate abasic scission of the second strand.
Cluster generation, linearization, blocking and sequencing primer reagents were provided in the Illumina Cluster Amplification kits. All samples are considered as biological replicates.
Sequencing was done on the Illumina Genome Analyzer IIx and on the Illumina HiSeq 2000. FastQ files for the resulting sequence reads (single read and paired-end, directional and non-directional) were moved to a data library in Galaxy, and tools implemented in Galaxy were used for further processing via workflows ((Giardine et al., 2005), (Blankenberg et al., 2010 ), (Goecks et al., 2010). Data processing was also performed on the CyberSTAR high-performance computing system at Penn State. The reads were mapped to the mouse genome (mm9 assembly) using the program TopHat ((Langmead et al., 2009) and (Trapnell et al., 2009)). Signal tracks were created using BEDtools ((Quinlan et al., 2010)) and SAMtools ((Li, Handasaker et al., 2009)).

first: Ross

last: Hardison

first: Robert

last: Paulson

first: David

last: Bodine

first: Mitchell

last: Weiss

first: Tejaswini

last: Mishra

first: Cheryl

last: Keller

first: Belinda

last: Giardine

first: James

last: Taylor


title: Otiwi1-interacting RNAs at four time points during Oxytricha conjugation

first: Wenwen

last: Fang

summary: Genome duality in ciliated protozoa offers a unique system to showcase their epigenome as a model of inheritance. In Oxytricha, the somatic genome is responsible for vegetative growth, while the germline contributes DNA to the next sexual generation. Somatic nuclear development eliminates all transposons and other "junk DNA", which constitute ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally-deleted regions leads to their retention in subsequent generations. Our findings highlight small RNAs as powerful transgenerational carriers of epigenetic information for genome programming.

overall_design:
We sequenced sRNAs enriched by Otiwi1 co-IP at 12, 19, 23 and 30 hr post-mixing during Oxytricha sexual development.
RNA ChIP with Otiwi1 antibody (Abcam, cat# ab12337, lot#868987) enriched 27nt sRNAs in Oxytricha at 12, 19, 23 and 30 hr post-mixing of mating types.


title: Genome-wide mapping of Olig2 targets in primary oligodendrocytes

first: Qing

middle: Richard

last: Lu

address: 5323 Harry Hines Boulevard

institute: UT southwestern medical center

summary: Myelination by oligodendrocytes in the central nervous system (CNS) is essential for proper brain function, yet the molecular determinants that control this process remain poorly understood. The basic helix-loop-helix transcription factors Olig1 and Olig2 promote myelination, whereas bone morphogenetic protein (BMP) and Wnt/β-catenin signaling inhibit myelination. Here we show that these opposing regulators of myelination are functionally linked by the Olig1/2 common target Smad-interacting protein-1 (Sip1). We demonstrate that Sip1 is an essential modulator of CNS myelination. Sip1 represses differentiation inhibitory signals by antagonizing BMP receptor-activated Smad activity while activating crucial oligodendrocyte-promoting factors. Importantly, a key Sip1-activated target, Smad7, is required for oligodendrocyte differentiation and partially rescues differentiation defects caused by Sip1 loss. Smad7 promotes myelination by blocking the BMP- and β-catenin-negative regulatory pathways. Thus, our findings reveal that Sip1-mediated antagonism of inhibitory signaling is critical for promoting CNS myelination and point to new mediators for myelin repair.

overall_design: ChIP-seq was performed to identify Olig2 direct target genes in oligodendrocytes during oligodendrocyte differentiation.

middle: R


title: Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals [RNA-seq]

first: Joseph

last: Takahashi

address: 5323 Harry Hines Blvd., NA4.118

department: HHMI, Neuroscience

institute: University of Texas Southwestern Medical Center

summary: The mammalian circadian clock involves a transcriptional feedback loop in which CLOCK and BMAL1 activate the Period and Cryptochrome genes, which then feedback and repress their own transcription. We have interrogated the transcriptional architecture of the circadian transcriptional regulatory loop on a genome scale in mouse liver and find a stereotyped, time-dependent pattern of transcription factor binding, RNA polymerase II (RNAPII) recruitment, RNA expression and chromatin states. We find that the circadian transcriptional cycle of the clock consists of three distinct phases — a poised state, a coordinated de novo transcriptional activation state, and a repressed state. Interestingly only 22% of mRNA cycling genes are driven by de novo transcription, suggesting that both transcriptional and post-transcriptional mechanisms underlie the mammalian circadian clock. We also find that circadian modulation of RNAPII recruitment and chromatin remodeling occurs on a genome-wide scale far greater than that seen previously by gene expression profiling.

overall_design: Examination of whole transcriptome every 4hr during the circadian cycle in mouse liver

first: Nobuya

last: Koike

first: Seung-Hee

last: Yoo

first: Hung-Chung

last: Huang

first: Vivek

last: Kumar

first: Choogon

last: Lee

first: Tae-Kyung

last: Kim


title: POLIISER5 and POLIISER2 ChIP-Seq in mutant RNAi LID Drosophila Melanogaster

city: Barcelona

last: Rossell

address: Baldiri Reixac 10

institute: IRB Barcelona

summary: LID is a histone demethylase acting on H3K4me3, a mark related to transcription and found near the transcription start sites (TSS) of the genes. We analyzed where POLIISER5 and POLIISER2 are localized in LID RNAi mutants.

overall_design: 1 sample for POLIISER5 with an input control, and 1 sample for POLIISER2 with an input control.

first: Ferran

last: Azorin