title: Natural genetic variation perturbs collaborative transcription factor binding required for enhancer selection and function

first: Casey

last: Romanoski

address: 9500 Gliman Dr.

summary: Natural genetic variation between two mouse strains was used as a natural mutagenesis screen to test various aspects of the proposed model for enhancer selection and function.

overall_design: Chromatin marks H3K4me2, H3K27Ac, and transcription factors PU.1, C/EBPa, and p65 were assessed by ChIP-Seq and GRO-Seq and RNA-Seq were used to measure gene expression in C57BL6/J and BALBc/J inbred mouse strains

first: Christopher


title: Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation [ChIP-Seq]

status: Public on Oct 11 2013

city: Boston

first: Sebastien

last: Vigneau

state: MA

address: 450 Brookline Avenue

department: Cancer Biology

country: USA

institute: Dana-Farber Cancer Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: CTCF (CCCTC-binding factor) is a highly conserved 11-zinc finger DNA binding protein with tens of thousands of binding sites genome-wide. CTCF acts as a multifunctional regulator of transcription, having been previously associated with activator, repressor, and insulator activity. These diverse regulatory functions are crucial for preimplantation development and are implicated in the regulation of numerous lineage-specific genes.  Despite playing a critical role in  developmental gene regulation, the mechanisms that underlie developmental changes in CTCF recruitment and function are poorly understood. Our previous work suggested that differences in CTCF’s binding site sequence may affect the regulation of CTCF recruitment, as well as CTCF’s regulatory function. To investigate these two possibilities directly during a developmental process, changes in genome-wide CTCF binding and gene expression were characterized during in vitro differentiation of mouse embryonic stem cells. CTCF binding sites were initially separated into three classes (named LowOc, MedOc, and HighOc) based on similarity to the consensus motif.  The LowOc class, with lower-similarity to the consensus motif, is more likely to show changes in binding during differentiation.  These more dynamically bound sites are enriched for motifs that confer a lower in vitro affinity for CTCF, suggesting a mechanism where sites with low-binding affinity are more amenable to developmental control. Additionally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions.  In sum, these results suggest that the regulatory control of CTCF’s binding and function is dependent in part upon specific motifs within its DNA binding site.

overall_design: Mouse E14 ES cells were differentiated in vitro for 4.5 days using retinoic acid. ChIP-seq for CTCF and an IgG control was performed from cells collected before and after differentiation. For undifferentiated cells, data were generated in two biological replicates.

first: Robert

middle: N

last: Plasschaert

first: Sébastien

first: Italo

last: Tempera

first: Ravi

last: Gupta

first: Jasna

last: Maksimoska

first: Logan

last: Everett

first: Ramana

last: Davuluri

first: Ronen

last: Mamorstein

first: Paul

middle: M

last: Lieberman

first: David

last: Schultz

first: Sridhar

last: Hannenhalli

first: Marisa

middle: S

last: Bartolomei


title: Allele-specific Methylation of Human Placental Tissue

status: Public on Oct 09 2013

city: Wenzhou

first: Chunming

last: Ding

state: Zhejiang

address: School of Laboratory Medicine and Life Sciences

country: China

institute: Wenzhou Medical University

type: Methylation profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

summary: In this study, we screened human placental samples for allele-specific methylation and subsequently novel imprinted genes associated with these regions. We used reduced representation bisulfite sequencing to identify partially methylated CpG islands (CGIs) in the human placental genome. We were able to delineate potential candidates for allele-specific methylation based on the calculation of a concordance statistic. Amongst the 28 regions chosen for validation based on high levels of expression, two regions were shown to exhibit allele-specific expression.

overall_design: Single base-resolution methylation analysis in the placental genome and RNA-Seq


title: Identification of miR-4423 as a Primate-Specific microRNA Highly Expressed in Ciliated Airway Epithelium and Associated with Lung Cancer

status: Public on Oct 07 2013

first: Avrum

last: Spira

address: 72 East Concord Street, E631

department: Computational Biomedicine

institute: Boston University

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Transcriptome analysis of the Zebrafish model of Diamond-Blackfan Anemia from RPS19 deficiency via p53-dependent and -independent pathways

city: Beijing

first: yadong

last: yang

address: No.1-104 Beichen West Road, Chaoyang

institute: Beijing Institute of Genomics

summary: To determine the effect on the whole transcriptome of RPS19 morpholino and to identify the function of p53 in RPS19-deficient embryos, we analyzed the genome-wide transcript profiles of control morpholino (control), RPS19 morpholino knockdown (RPS19 MO), and RPS19 and p53 morpholino knockdown simultaneously (RPS19+p53 MO) using the Illumina Hi-seq 2000 Genome Analyzer platform with paired-end 100 base-pair tags to a depth of 35-60 million reads. We found that genes enriched in the functions of hematological systems and nervous system development and that skeletal and muscular disorders had significant differential expression in RPS19 MO embryos compared with controls. Co-inhibition of p53 partially alleviates the abnormalities for RPS19-deficient embryos. However, the hematopoietic genes, which were down-regulated significantly in RPS19 MO embryos, were not completely recovered by the co-inhibition of p53. Furthermore, we identified the genome-wide p53-dependent and -independent genes and pathways. These results indicate that not only p53 family members but also several other factors have important impacts on RPS19-deficient embryos. The detection of potential pathogenic genes and pathways provides us a new paradigm for research on DBA, which is a systematic and complex hereditary disease.

overall_design: Compare mRNA transcriptomes of three different souces of zebrafish embryos

first: Xiangdong

last: Fang


title: Extensive Variation in Chromatin States Across Humans

status: Public on Oct 18 2013

city: Stanford

first: Maya

last: Kasowski

address: 300 Pasteur Drive

institute: Stanford University

summary: The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans.

overall_design: To characterize human variation in diverse types of regulatory elements, we studied the chromatin state of lymphoblastoid cell lines (LCLs) derived from 19 individuals: 5 European (CEU), 7 Yoruban (YRI), and 2 Asian individuals from the 1000 Genomes Project (including two mother-father-daughter trios), an additional Caucasian individual), and four individuals from the San population). We used RNA-Seq to measure expression and Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) to map five histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, and H3K27me3) and two general factors (CTCF, and SA1, a subunit of cohesin). We generated 2 replicates per factor.

first: Sofia

last: Kyriazopoulou-Panagiotopoulou

first: Fabian

last: Grubert

first: Judith

last: Zaugg

first: Anshul

last: Kundaje

first: Yuling

last: Liu

first: Alan

middle: P

last: Boyle

first: Qiangfeng

middle: C

last: Zhang

first: Fouad

last: Zakharia

first: Damek

middle: V

last: Spacek

first: Jingjing

last: Li

first: Dan

last: Xie

first: Anthony

last: Olarerin-George

first: Lars

last: Steinmetz

first: John

middle: B

last: Hogenesch

first: Manolis

last: Kellis

first: Serafim

last: Batzoglou

first: Michael

last: Snyder


title: The Wnt/β-catenin-signaling pathway is modulated by androgen ablation therapy for advanced clinical prostate cancer and contributes to androgen independent cell growth

city: Sheffield

first: Ian

last: Sudbery

address: Firth Court, Western Bank

department: Molecular Biology and Biotechnology

country: United Kingdom

institute: University of Sheffield

summary: Androgen ablation therapy (AAT) is standard treatment for locally-advanced/metastatic prostate cancer (PCa).  Many patients develop castration-resistance (CRPCa) after ~2-3 years, with a poor prognosis.  The molecular mechanisms underlying CRPCa progression are unclear.  mRNA-Seq was performed on tumours from 7 patients with locally-advanced/metastatic PCa before and ~22 weeks after AAT initiation.  Differentially regulated genes were identified in treatment pairs.

overall_design: Tumour biopsies from 7 patients were taken before and after AAT treatment

first: Prabhaker

last: Rajan

first: M

last: Villasevil

first: Ernest

last: Mui

first: Janis

last: Fleming

first: Mark

last: Davis

first: Albiruni

middle: R

last: Abdul Razak

first: Imran

last: Ahmad

first: Joanne

last: Edwards

first: Owen

middle: J

last: Sansom

first: David

last: Sims

first: Chris

last: Ponting

first: Andreas

last: Heger

first: Rhona

last: McMenemin

middle: D

last: Pedley

first: Hing

middle: Y

last: Leung


title: Genome-wide identiﬁcation of Vitis vinifera microRNAs reveals their differential regulation by gibberellin

status: Public on Oct 05 2013

city: Nanjing

first: Changnian

last: Song

address: Weigang No.1

department: College of Horticulture

institute: Nanjing Agricultural University

summary:
MicroRNAs (miRNAs) play a important part in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes.  There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice and other plants.  However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination.  In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology.  A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered.  Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine.  QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and  26 miRNAs being specifically induced by gibberellin exposure.  All novel sequences had not been earlier described in other plant species.  In addition, 117 target genes for 29 novel miRNAs were successfully predicted.  Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries.  This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries.
GSM604831 is the control for the gibberellin-treated sample.

overall_design: The mixture samples of young berries (one week after flowering) large berries (five week after flowering after flowering), and old berries (nine week after flowering) treated with gibberellin, respectively, were generated by deep sequencing, in triplicate, using Illumina 1G Genome Analyzer.

first: J

last: Han

first: C

last: Fang


title: Dynamic transcriptomes of human myeloid leukemia cells

first: yadong

last: yang

summary: To identify the mechanisms controlling chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) in humans, we analyzed genome-wide transcription dynamics in three myeloid leukemia cell lines (K562, HL-60, and THP1) using high-throughput sequencing technology. Using KEGG analysis, we found that the ERK/MAPK, JAK-STAT and ErbB pathways promoted proliferation and metabolism in CML. However, in AML, differentiation and apoptosis blocking resulted in the accumulation of blast cells in marrow. In addition, each cell type had unique characteristics. K562 cells are an ideal model for studying erythroid differentiation and globin gene expression. The chemokine signaling pathway and Fc gamma R-mediated phagocytosis were markedly upregulated in HL-60 cells. In THP1 cells, highly expressed genes ensured strong phagocytosis by monocytes. Further, we provide a new insight into myeloid development. The abundant data sets and well-defined analysis methods will provide a resource and strategy for further investigation of myeloid leukemia.

overall_design: Compare mRNA transcriptomes of three different cell lines

first: Xiangdong

first: Songnian

last: Hu


title: TRIM28 is essential for erythroblast differentiation in the mouse

status: Public on Oct 04 2013

city: Ann Arbor

first: Tomonori

last: Hosoya

state: MI

address: 109 Zina Pitcher Place

department: CDB

institute: University of Michigan

summary: We discovered that Trim28 genetic loss in the adult mouse leads to defective immature erythropoiesis in the bone marrow and consequently to anemia.We further found that TRIM28 controls erythropoiesis in a cell-autonomous manner by inducibly deleting Trim28 exclusively in hematopoietic cells. Finally, in the absence of TRIM28 we observed increased apoptosis as well as diminished expression of multiple erythroid transcription factors and heme biosynthetic enzymes in immature erythroid cells. Thus, TRIM28 is essential for the cell-autonomous development of immature erythroblasts in the bone marrow.

overall_design: To induce Cre recombinase from the Mx1Cre transgene, poly(I:C) was injected 5 times every other day. Mice were analyzed two weeks after completion of poly(I:C) administration. TRIM28 mutant mice generate two distinct types of immature erythroid cells; KOa, with 5-8% of the Trim28 gene remaining undeleted (still bearing 26% of residual mRNA), and KOb, with 1-4% of the gene remaining undeleted (and with 18% of mRNA remaining).

first: James

last: Engel


title: Identification of miR-4423 as a Primate-Specific microRNA Highly Expressed in Ciliated Airway Epithelium and Associated with Lung Cancer [high throughput sequencing]

status: Public on Oct 08 2013

first: Avrum

last: Spira

summary: Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. While microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify miR-4423 as a novel primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a novel regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis.

overall_design: Small RNA expression was profiled from pooled bronchial airway epithelial cell brushings (n=3 patients/pool) obtained during bronchoscopy from healthy never (NS) and current smokers (S) and smokers with (C) and without (NC) lung cancer. MicroRNA hsa-miR-4423 was over expressed in H1299, Calu6, SW900 and H2170 lung cancer cell lines.

first: Catalina

last: Perdomo

first: Joshua

last: Campbell

first: Joseph

last: Gerrein

first: Carmen

last: Tellez

first: Carly

last: Garrison

first: Tonya

last: Walser

first: Eduard

last: Drizik

first: Huiqing

last: Si

first: Adam

last: Gower

first: Jessica

last: Vick

first: Christina

last: Anderlind

first: George

last: Jackson

first: Courtney

last: Mankus

first: Frank

last: Schembri

first: Carl

last: O’Hara

first: Brigitte

last: Gomperts

first: Steven

last: Dubinett

first: Patrick

last: Hayden

middle: A

last: Belinsky

first: Marc

middle: E

last: Lenburg


title: Deep sequencing of grapevine flower and berry short RNA library for discovery of novel microRNAs and validation of precise sequences of grapevine microRNAs deposited in miRBase

first: Chen

last: Wang

state: Jiangshu

address: Weigang 1#

department: Horticulture College

institute: Nanjing Agriculture University

summary: MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms.  The advent of new high throughput sequencing technologies has enabled the revelation of novel miRNAs.  Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were significant divergence in the number and kinds of miRNAs sequenced in these studies.  More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine.  Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar ‘Summer Black’ and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries.  Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered.  Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity.  130 target genes for 46 novel miRNAs could be predicted.  The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed.

overall_design: Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.


title: Genome-wide analysis of condensin binding sites in vertebrate genome

status: Public on Oct 03 2013

city: Parkville

first: Ji Hun

last: Kim

state: VIC

address: Royal Childrens Hospital, 50 Flemington Rd

department: Paediatrics

country: Australia

institute: Murdoch Childrens Research Institute / The Univesity of Melbourne

summary: The condensin complex is well known to be essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes. To date, the genome wide location and the nature of condensin binding sites has remained elusive in vertebrate systems. A detailed knowledge of condensin binding sites is essential to understand the as-yet enigmatic function of this complex and to define its greater role in chromosome architecture. To address this, we have used next generation sequencing to map condensin I in chicken DT40 cells. Unexpectedly, we found condensin I binds predominately to promoter sequences in mitotic cells. We also found a striking enrichment at both centromeres and telomeres, highlighting the importance of the complex in chromosome segregation. Taken together, the results show condensin I is largely absent from heterochromatic regions. These results reveal for the first time the condensin I binding sites in the vertebrate genome, and demonstrate the importance of the complex in genome segregation and suggest a novel function in gene regulation.

overall_design: Condensin associated DNA from chicken DT40 cells were affinity purified using streptavidin and deep sequenced by Illumina Hiseq2000

first: Ji

middle: H

first: Damien

middle: F

last: Hudson


title: High throughput sequencing enables detailed analysis of S. cerevisiae DNA  replication

status: Public on Oct 02 2013

city: Norwich

first: Conrad

last: Nieduszynski

address: Norwich Research Park

institute: Earlham Institute

summary: We have used three complementary deep sequencing approaches to characterise the temporal order of genome replication in S. cerevisiae. Each approach measures the increase in DNA copy number as a genomic region is replicated (in a large population of cells). We find that the use of deep sequencing provided high spatial resolution. For maximum temporal resolution we have measured replication dynamics at multiple time points during a synchronous S phase. To pinpoint replication origins we have synchronously released a checkpoint mutant (rad53-delta) into S phase under conditions of depleted dNTPs (200 mM hydroxyurea; HU) and measured the increase in DNA copy number after 60 minutes. Finally for rapid characterisation of replication dynamics in wild-type cells from an unperturbed cell cycle we have obtained replicating (S phase) and non-replicating (G2 phase) cells by fluorescence activated cell sorting (FACS) and subjected the DNA to copy number analysis. We have compared and contrasted these approaches and developed the tools required for interpreting the deep sequencing data.

overall_design: Measurement of genome replication time for various S. cerevisiae strains. For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase).

first: Carolin

last: Müller

first: Michelle

last: Hawkins

first: Renata

last: Retkute

first: Martin

last: Blythe

first: Sunir

last: Malla

first: Raymond

last: Wilson


title: Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

city: Gainesville

first: Nancy

last: Nabilsi

state: FL

address: 2033 Mowry Rd

department: Biochemistry and Mol Bio

institute: University of Florida

summary: Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from sub-microgram input quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintains the structural integrity of epigenetic information and provides substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. Additionally, substantial epigenetic heterogeneity was observed across the sequenced molecules at individual promoters, indicating the presence of epigenetically distinct cellular subpopulations.

overall_design: This study includes 4 samples, NSC probed with 100U M.CviPI and 0U control; GBM probed with 100U M.CviPI and 0U control.

first: Russell

last: Darst

first: Michael

last: Kladde


title: PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G+C-rich regions of the mouse genome.

city: Montpellier

last: Girardot

state: France

address: 1919 route de Mende

country: France

institute: IGMM CNRS UMR5535

summary: Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

sample_organism: Mus musculus domesticus x M. m. molossinus

overall_design: High throughput sequencing data from H4R3me2s native ChIP samples  from mouse embryonic stem cells and fibroblasts were generated using Illumina Hi-seq 2000.

first: Robert

last: Feil


title: De Novo Purine Synthesis Mediates Circadian Control of Cell Cycle in Zebrafish (HTS)

status: Public on Oct 01 2013

city: Shanghai

first: Guang

last: Li

address: 320 Yueyang Lu

institute: PICB

summary: Inosine 5'-phosphate dehydrogenase (impdh) has been well known as a key enzyme in GTP biosynthesis pathway. We found that three isoforms of impdh in zebrafish, namely impdh1a, impdh1b and impdh2, all show robust circadian expression.To examine the molecular functions of three impdh isoforms in zebrafish on the genome scale, we measured the global expression changes of impdh1a, impdh1b and impdh2 morpholino injected larvae (morphants) respectively using RNA-seq. Wild type (WT), control and three impdh morphants were collected at 32 hpf. In our RNA-seq result, we identified 468, 331 and 1166 significant genes affected by impdh1a, impdh1b and impdh2 morpholino (MO) knock-down respectively. Among them, there are limited overlaps between genes affected by different MOs and only 36 genes in common among all three MOs. This indicates that the three impdh isoforms have distinct molecular functions.

overall_design: To knock down the target genes, three impdh MOs and control MO were pressure-injected into 1- to 2-cell stage embryos. WT, control and three impdh morphants were raised at 28°C under 14h: 10h light/dark cycle from birth and sampled simultaneously at 32 hpf.  Each group has at least 40 embryos.

first: Y

first: G

first: J

last: Du

last: Yan


title: ChIP-seq of MYST acetyltransferases

city: Quebec city

first: Marie-Eve

last: Lalonde

state: Qc

address: 9 McMahon street

country: Canada

institute: CHUQ

summary: Histone acetyltransferases (HAT) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones.  Here, we characterize native HAT complexes assembled by the BRPF-family of scaffold proteins.  Their PHD-ZnKnuckle-PHD domain is essential for binding chromatin and restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit.  Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit.  Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin.  We mapped a small region at the N-terminus of scaffold proteins responsible for histone tail selection on chromatin.  Thus, alternate choice of subunits associated with HBO1 can switch its specificity from H4 to H3 tails, highlighting a crucial new role of associated subunits within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

overall_design: Genome-wide mapping of MYST acetyltransferases subunits and H3K4me3 histone mark in RKO cells.

first: Eric

last: Paquet

first: Jacques

last: Côté


title: Identification of genetic variants that affect histone modifications in human cells

city: La Jolla

first: Graham

last: McVicker

state: CA

address: 10010 N Torrey Pines Rd

institute: Salk Institute

summary: Histone modifications are important markers of function and chromatin state, yet the DNA elements that direct them to specific locations in the genome are poorly understood. Here we use the genetic variation in Yoruba lymphoblastoid cell lines as a natural experiment to identify genetic differences that affect histone marks and to better understand their relationship with transcriptional regulation. Across the genome, we identified hundreds of quantitative trait loci that impact histone modification or RNA polymerase (PolII) occupancy. In many cases the same variant is associated with quantitative changes in multiple histone marks and PolII, as well as in DNaseI sensitivity and nucleosome positioning, indicating that these molecular phenotypes often share a single underlying genetic cause.  Variants that impact chromatin at distal regulatory sites frequently also direct changes in chromatin and gene expression at associated promoters; while most of these distal regulators enhance promoter activity, some act as distal chromatin silencers. Finally, we find that polymorphisms in transcription factor binding sites are often causally responsible for variation in local histone modification.  In summary, the class of variants identified here generate coordinated changes in chromatin both locally and sometimes at distant locations, frequently drive changes in gene expression, and likely play an important role in the genetics of complex traits.

overall_design: ChIP-seq of RNA Polymerase II and 4 histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3) in 10 unrelated Yoruba HapMap lymphoblastoid cell lines

first: Bryce

last: van de Geijn

first: Jacob

last: Degner

first: Carolyn

last: Cain

first: Nicholas

last: Banovich

first: Anil

last: Raj

first: Noah

last: Lewellen

first: Marsha

last: Myrthil

first: Yoav

last: Gilad

first: Jonathan

middle: K

last: Pritchard


title: De Novo Purine Synthesis Mediates Circadian Control of Cell Cycle in Zebrafish

first: Guang


first: Vanessa

middle: K.

last: Ridaura

state: MO

address: 4444 Forest Park Parkway, Box 8510

department: Pahtology and Immunology

institute: Washington University in St. Louis, School of Medicine

summary: Microbial RNAseq analysis of cecal and fecal samples collected from mice colonized with the microbiota of human twins discordant for obesity. Samples were colleted at the time of sacrifice, or 15 days after colonization from mice gavaged with uncultured or cultured fecal microbiota from the lean twins or their obese co-twins. Samples were sequenced using Illumina HiSeq technology, with 101 paired end chemistry.

overall_design: Comparisson of microbial gene expression between the microbiota of lean and obese twins fed a Low fat, rich in plant polysaccharide diet.


title: POLR3G and POLR3GL-RNA polymerase III target genes

city: Lausanne

first: Nicolo

last: Riggi

state: VD

address: Bugnon 25

department: Département de Pathologie Expérimentale

country: Switzerland

institute: CHUV

summary:
Purpose:  Characterization of the target genes of two versions of the Polymerase III (pol III), one containing POLR3G subunit or POLR3GL subunit, in different tissues.
Methods: We performed ChIP_seq of POLR3D and the two variants subunits, POLR3G and POLR3GL in Human IMR90 cells, Mouse liver and Mouse Hepatocarcinoma Hepa 1-6 cells in order to locate and analyse total pol III, POLR3G and POLR3GL-containing pol III in various tissues.
Results: We show that POLR3G- as well as POLR3GL-containing pol III are present in cultured cell lines and in normal mouse liver, although the relative amounts of the two forms vary, with the POLR3G-containing pol III relatively more abundant in dividing cells. Both forms of pol III occupy the same target genes, in very constant proportions within one cell line, suggesting that the two forms of pol III have similar function with regard to specificity for target genes.
Conclusions: POLR3G and POLR3GL occupy the same target genes, but they are differentially expressed under different conditions and in different cell types.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Pol III target genes in human IMR cells, Mouse liver, and Mouse Hepatocarcinoma cells were identified by chromatin Immuno-precipitation followed by deep sequencing using Illumina HiSeq. For Human IMR90 cells, we used antibodies against POLR3D, POLR3G, POLR3GL and BDP1. For Mouse data, we targeted POLR3D, POLR3G and POLR3GL and did ChIP_seq replicates. We also sequenced the corresponding inputs (crosslinked DNA of IMR90 cells, Mouse liver, and Mouse Hepatocarcinoma cells). This represents a total of 20 samples.

first: Marianne

last: Renaud

first: Viviane

last: Praz

first: Erwann

last: Vieu

first: Laurence

last: Florens

first: Michael

last: Washburn

first: Philippe

last: L'Hôte

first: Nouria

last: Hernandez


title: Developmental establishment of DNA methylation in hypothalamic neurons and glia.

city: Houston

first: Ge

last: Li

state: TX

address: 1100 Bates St.

department: Pediatrics

institute: Baylor College of Medicine

summary: Human epidemiologic and animal model data indicate that early environmental influences can persistently alter an individual’s risk of obesity. Environmental effects on hypothalamic developmental epigenetics provide a strong candidate mechanism to explain such ‘developmental programming’ of obesity. To advance our understanding of these processes, it is essential to determine to what extent the diversity of hypothalamic cell types is regulated by epigenetic differences, and when these are established. By performing genome-scale DNA methylation profiling in hypothalamic neurons and non-neuronal cells at postnatal day 0 (P0) and P21, we found that most of the DNA methylation differences distinguishing these two cell types are established postnatally. We found dramatic neuron-specific increases in DNA methylation from P0 to P21. Gene ontology analyses indicated that cell-type specific P0 to P21 methylation changes are key regulators of hypothalamic development. Quantitative bisulfite pyrosequencing verified our methylation profiling results in 16 of 16 selected regions. Expression differences were associated with DNA methylation in several genes analyzed. Our data indicate that future studies of hypothalamic epigenetics in developmental programming of obesity will gain far greater sensitivity and insight by examining outcomes at the cell-type specific level.  Moreover, our results provide new evidence that early postnatal life is a critical period for murine hypothalamic developmental epigenetics.

overall_design: Hypothalami were dissected from inbred male C57 mice at postnatal day 0 (P0) and P21. Non-neuronal and neuronal nuclei were separated via fluorescence-activated sorting based on staining for the neuron-specific nuclear surface marker NeuN; each sample for sorting was comprised of 2 age-matched hypothalami. Genome-scale DNA methylation profiling was performed by methylation specific amplification coupled with next generation sequencing (MSA-seq) as decribed below (5 independent samples per age).

first: Govindarajan

last: Ramamoorthy

first: Robert

last: Waterland


title: DNA methylation in hypothalamus

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

overall_design: Refer to individual Series


title: Retroelements and DUX4 Create Primate-specific Promoters for Germline Genes

city: Seattle

first: Stephen

last: Tapscott

state: WASHINGTON

address: 1100 Fairview N. Ave

department: Human Biology

institute: Fred Hutch Cancer Research Center

summary: The human double-homeodomain retrogene DUX4 is normally expressed at high levels in germ cells of the testis. When aberrantly expressed in muscle its protein product causes facioscapulohumeral muscular dystrophy (FSHD), perhaps partly by inducing inappropriate expression of germline genes. DUX4 can bind >60,000 locations in the human genome that contain a strongly enriched sequence motif. Numerous long terminal repeat (LTR) class repetitive elements are enriched among DUX4 binding sites, including many from the mammalian apparent LTR-retrotransposon (MaLR) family as well as some ERVL and ERVK types, with MaLRs comprising ~1/3 of DUX4’s binding sites. We performed RNA-seq on myoblasts over-expressing DUX4 and find that DUX4 binding activates transcription of some but not all bound repeat types. Some of these activated repetitive elements comprise novel promoters for genes, long non-coding RNAs and antisense transcripts. We show that some of these chimeric repeat-initiated transcripts are expressed in testis and FSHD patient myotubes. The acquisition of MaLR-LTR elements during mammalian evolution may therefore have allowed rewiring of the transcriptional network. We also find that the pericentromeric satellite HSATII can be bound by DUX4 and that its transcription is massively induced by DUX4 over-expression. Our findings suggest a role for repetitive element transcripts in muscle disease and in the biology of normal testis.

overall_design: RNA-seq of two myoblast cell lines transduced with lentivirus carrying DUX4, and two control myoblast lines

first: Janet

last: Young

first: Jennifer

middle: L

last: Whiddon

first: Zizhen

last: Yao

first: Bhavatharini

last: Kasinathan

first: Lauren

last: Snider

first: Linda

middle: N

last: Geng

first: Rabi

last: Tawil

first: Silvere

last: van der Maarel


title: Genome-wide total RNA and MBD-sequencing in HCT116 and DKO cells as a global re-expression model

city: Gent

first: Joachim

last: De Schrijver

address: Coupure Links 653

country: Belgium

institute: Universiteit Gent


title: Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

first: William

last: Greenleaf

address: 279 Campus Dr West, Beckman Center

department: Genetics

institute: Stanford University

summary: We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500–50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with the nucleosome. Using ATAC-seq maps of human CD4+ T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual’s epigenome on a timescale compatible with clinical decision-making.

overall_design: We examined chromatin structure using ATAC-seq in 2 cell types (GM12878 cell line, purified CD4+ T cells).

first: Jason

last: Buenrostro

first: Paul

middle: G

last: Giresi

first: Lisa

last: Zaba

first: Howard

last: Chang


title: Genome-wide total RNA and MBD-sequencing in HCT116 and DKO cells as a global re-expression model [RNA-Seq]

summary: Genome-wide view of the interplay between methylation and RNA expression in this colorectal cancer model was obtained.

overall_design: RNA-sequencing of HCT116 cell line, the DNMT1 and DNMT3B double knock-out HCT116 cell line (DKO), and 7 additional colorectal cell lines.

first: Geert

last: Trooskens

first: Linda-J

middle: W

last: Bosch

first: Pierre

last: Dehan

first: Beatriz

last: Carvalho

first: Leander

last: Van Neste

first: Gerrit

last: Meijer

first: Steve

last: Baylin

first: Wim

last: Van Criekinge


title: Genome-wide total RNA and MBD-sequencing in HCT116 and DKO cells as a global re-expression model [MBD-Seq]

overall_design: MBD-sequencing of 8 colorectal cancer cell lines.


title: Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia

first: Mien-Chie

last: Hung

address: 1515 Holcombe Boulevard

institute: MD Anderson Cancer Center


title: Next-Generation Sequencing for Whole Transcriptome Analysis in Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia

summary:
Purpose: The goal of this study is to identify the mRNA clusters that are regulated by EGFR under normoxia or hypoxia.
Method: Total RNAs were extracted from HeLa cells expressing scrambled control or EGFR shRNA-E1 that cultured under normoxia or hypoxia (1% O2) for 24h. Customized Next-Generation RNA Deep Sequencing, including both small RNA application and whole transcriptome analysis, was performed according to the standard procedure instructed by Applied Biosystems. For whole transcriptome analysis, SOLiD fragment colorspace transcriptome reads (50nt) were mapped to the human genome (hg19) and assigned to ensemble transcripts using Bioscope 1.3.1 (Life Technologies). The values of reads per kilobase per million reads (RPKM) were determined by Bioscope 1.3.1 CountTags tool using default parameters. Primary alignments with a minimum mapping quality of 10 and minimum alignment score of 10 were counted.
Results: Deep sequencing analysis identified subclasses of mRNAs that were affected by EGFR either under normoxia or hypoxia. EGFR-regulated mRNAs (with Log2 fold-change affected by EGFR ≥ 0.4 or ≤ -0.4) were sorted and over-lapped with mRNAs that were targeted (based on published data and TargetScan prediction with total context score ≤ -0.20) by the top miRNA candidates affected by EGFR under hypoxia, resulting in 439 mRNAs that regulated by EGFR and likely targeted by the miRNA candidates in response to hypoxia.
Conclusion: Whole transcriptome analysis revealed a novel cluster of mRNAs that are likely regulated by EGFR through miRNAs in response to hypoxic stress.

overall_design: RNA profiles of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) that cultured under normoxia or hypoxia (1% O2) for 24h were generated by AB SOLiD curstomarized next-generation sequencing, including both small RNA application and whole transcriptome analysis. S: HeLa expressing scrambled control cultured under normoxia; A1: HeLa expressing EGFR shRNA-E1 cultured under normoxia; HS: HeLa expressing scrambled control cultured under hypoxia for 24h; HA1: HeLa expressing EGFR shRNA-E1 cultured under hypoxia for 24h. In total, 4 biological samples with no replicates resulted in 4 whole transcriptome RNA profiles.

first: Jia

last: Shen

first: Brian

middle: P

last: James

first: Xiuping

last: Liu

first: Chang-Gong


title: Quantification of gene expression from primate tissue samples

city: Luebeck

first: Martin

last: Kircher

state: SH

address: Ratzeburger Allee 160

department: Humangenetics

country: Germany

institute: University of Luebeck

summary:
We have quantified gene expression in five tissues (brain, heart, kidney, liver and testis) from humans, chimpanzees and rhesus macaques using the Illumina NlaIII Digital Gene Expression (DGE) protocol. This dataset extends a previous microarray study by Khaitovich et al. (Khaitovich et al. 2005) with the rhesus macaque outgroup and complements other previously generated tissue transcriptome profiles from primates (Enard et al. 2002; Khaitovich et al. 2006; Somel et al. 2009; Babbitt et al. 2010; Blekhman et al. 2010; Wetterbom et al. 2010).
contributor:
Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens

overall_design: Samples were obtained from brain (pre-frontal cortex), heart, kidney, liver, and testis tissues of male humans, chimpanzees and rhesus macaques. Illumina NlaIII DGE libraries for all samples were generated in tissue batches, randomizing species in library preparation and sequencing. Human samples originate from different, probably unrelated, individuals for each tissue. For chimpanzees and rhesus macaques the libraries for all tissues come from the same set of individuals and among these are individuals related at the half- and full-sibling level. Due to limited access to samples, the analysis could not be limited to individuals of similar age. Human individuals vary between 5 and 88 years of age, chimpanzees between 6 years and 35 years of age and rhesus macaques between 3 and 9 years of age.


title: Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the eukaryotic steady state transcriptome

city: New York

first: Andre

last: Kahles

state: New York

address: 1275 York Avenue

department: Computational Biology Center

institute: Memorial Sloan-Kettering Cancer Center

summary: Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes aberrant transcripts arising from mutations and transcriptional mistakes. Moreover, differential transcript processing such as alternative precursor mRNA splicing (AS) can generate NMD substrates, however, the extent of coupled AS and NMD remained unclear. To investigate NMD targeting of AS variants, we performed transcriptome-wide splicing studies using Arabidopsis thaliana single and double mutants in the NMD factor homologues UPF1 and UPF3, as well as samples treated with the translation inhibitor cycloheximide (CHX). Our analyses revealed that at least 17.5% of all multi-exon, protein-coding genes produce splicing variants of all major types that are targeted by NMD, with a significant fraction originating from the splicing of cryptic introns. Furthermore, accumulation of many intron-retained mRNAs in the mutants, but not in response to CHX suggests the action of distinct routes of NMD with variable impact of translation. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. NMD-dependent AS variants are linked to diverse biological functions, including the poison exon-mediated regulation of signaling and posttranslational protein modification components. In addition to mRNAs, numerous non-coding RNAs as well as newly identified transcripts derived from intergenic regions were shown to be NMD responsive. In summary, our comprehensive analysis of AS-coupled NMD provides strong evidence for a major function of this pathway in shaping the transcriptome, having fundamental implications in gene regulation and quality control of transcript processing steps in higher eukaryotes.

overall_design: Comparison of gene expression and alternative splicing patterns in control and nonsense-mediated decay (NMD)-impaired Arabidopsis seedlings

first: Gabriele

last: Drechsel

first: Anil

last: Kesarwani

first: Eva

last: Stauffer

first: Jonas

last: Behr

first: Philipp

last: Drewe

first: Gunnar

last: Rätsch

first: Andreas

last: Wachter


title: The MLL3/MLL4 branch of the COMPASS family regulates H3K4 monomethylation at enhancers

status: Public on Sep 30 2013

city: Kansas City

first: Joan

last: Conaway

institute: Stowers Institute

summary: Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (COMplex of Proteins ASsociated with Set1) family from yeast to human and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which are found at transcription start sites in all eukaryotes. Our recent studies in Drosophila demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 and MLL4, are most closely related to Drosophila Trr. Here, we use ChIP-seq of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and find that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, we demonstrate that MLL4 is a major regulator of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, we found a redundant role between Mll3 and Mll4 in enhancer H3K4 monomethylation in mouse embryonic fibroblast  (MEF) cells. These findings suggest that mammalian MLL3/MLL4 function in the regulation of enhancer activity and enhancer-promoter communication during gene expression and that mutations of MLL3 and MLL4 found in cancer could exert their properties through enhancer malfunction.

overall_design: ChIP-Seq in mouse embryonic stem (mES) cells for MLL4. ChIP-seq of MLL4 and p300 in human parental HCT116 cells. ChIP-seq of H3K4me1, H3K4me2 and H3K4me3 in parental HCT116 cells  and HCT116 cells with Mll4∆set.

first: Deqing

last: Hu

first: Xin

last: Gao

first: Ali

last: Shilatifard


title: Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains

city: Tallahassee

first: Michelle

last: Arbeitman

state: Florida

address: 1115 W. Call Street

department: Medicine Biomedical Sciences

institute: Florida State University

summary: Drosophila melanogaster adult males perform an elaborate courtship ritual to entice females to mate. fruitless (fru), a gene that is one of the key regulators of male courtship behavior, encodes multiple male-specific isoforms (FruM). These isoforms vary in their carboxy-terminal zinc finger domains, which are predicted to facilitate DNA binding. By over-expressing individual FruM isoforms in fru-expressing neurons in either males or females and assaying the global transcriptional response by RNA-sequencing, we show that three FruM isoforms have different regulatory activities that depend on the sex of the fly.  We identified several sets of genes regulated downstream of FruM isoforms.

overall_design: RNA seqeuncing was performed on mRNA derived from adult male or female heads, for a total of 39 samples. These samples included two wild type genotypes (Berlin and Canton-S), two transheterozygous mutants for fru P1 (Df(3R)P14/Df(3R)fru4-40 and fruw12/ Df(3R)ChaM5), and 3 overexpressing genotypes (fru P1-Gal4: UAS-FruMA, UAS-FruMB, UAS-FruMC). There were at least 3 replicates from biological samples for all sex by genotype combinations.

first: J

last: Dalton

middle: M

last: Fear

first: S

last: Knott

first: B

last: Baker

first: L

last: McIntyre

first: M


title: Examining gene expression of the fungus Magnaporthe oryzae during challenge with the bacterium Lysobacter enzymogenes

city: Newark

first: Sridhara

last: Kunjeti

state: DE

address: 531, S. college Ave

department: Plant and Soil Sciences

institute: University of Delaware

summary: Plants and animals have evolved a first line of defense response to pathogens called innate or basal immunity. While basal defenses in these organisms are well studied, there is almost a complete lack of understanding of such systems in fungal species, and more specifically, how they are able to detect and mount a defense response upon pathogen attack. Hence, the goal of the present study was to understand how fungi respond to biotic stress by assessing the transcriptional profile of the rice blast pathogen, Magnaporthe oryzae, when challenged with the bacterial antagonist Lysobacter enzymogenes. Based on microscopic observations of interactions between M. oryzae and wild-type L. enzymogenes strain C3, we selected early and intermediate stages represented by time-points of 3 and 9 hours post-inoculation, respectively, to evaluate the fungal transcriptome using RNA-seq. For comparative purposes, we also challenged the fungus with L. enzymogenes mutant strain DCA, previously demonstrated to be devoid of antifungal activity. A comparison of transcriptional data from fungal interactions with the wild-type bacterial strain C3 and the mutant strain DCA revealed 463 fungal genes that were down-regulated during attack by C3; of these genes, 100 were also found to be up-regulated during the interaction with DCA. Functional categorization of genes in this suite included those with roles in carbohydrate metabolism, cellular transport and stress response. One gene in this suite belongs to the CFEM-domain class of fungal proteins. Another CFEM class protein called PTH11 has been previously characterized, and we found that a deletion in this gene caused advanced lesion development by C3 compared to its growth on the wild-type fungus.  We discuss the characterization of this suite of 100 genes with respect to their role in the fungal defense response.

overall_design: Included in the analysis are two biological replicates each of the fungus in buffer alone (control), the fungus challenged with wild type bacterium at 3 and 9 hours post-inoculation (hpi) and the fungus challeged with the mutant bacterium (mutated for ability to secrete antibiotics and enzymes) at 3 and 9 hpi.

first: Sandra

last: Mathioni

first: Nrupali

last: Patel

first: Bianca

last: Riddick

first: James

last: Sweigard

first: Kirk

last: Czymmek

first: Jeffrey

last: Caplan

first: Saritha

first: Vidhyavathi

last: Raman

first: Donald

last: Kobayashi

first: Nicole

last: Donofrio


title: Genome-wide analysis of BRCA1 and PALB2

city: Miami

first: Alessandro

last: Gardini

address: 1501 NW 10 Avenue

department: Sylvester Comprehensive Cancer Center

institute: University of Miami


title: Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells [RNA-Seq]

city: Dallas

first: Laura

last: Banaszynski

address: 5323 Harry Hines Blvd

department: Green Center for Reproductive Biology Sciences

institute: UT Southwestern Medical Center

summary: Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions coupled to incorporation of the histone variant H3.3. Here we show in mouse embryonic stem cells (ESCs) that H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. These promoters show reduced dynamics as determined by deposition of de novo synthesized histones, associated with reduced PRC2 occupancy. H3.3-depleted ESCs show upregulation of extraembryonic trophectoderm, as well as misregulation of other developmental genes upon differentiation. Our data demonstrate the importance of H3.3 incorporation in ESCs and suggest that changes in chromatin dynamics in its absence lead to misregulation of gene expression during differentiation. Moreover, our findings lend support to the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an “active” chromatin state.

overall_design: RNA-seq analysis of three embryonic stem cell lines (control, H3.3 KD1, and H3.3 KD2)

first: Scott

last: Dewell

first: Deyou

last: Zheng

first: C

last: Allis


title: A next generation sequencing approach to profiling the host miRNA response to a bovine mastitis pathogen

city: Meath

first: Nathan

last: Lawless

address: Grange

country: Ireland

institute: Teagasc

summary: Substantial evidence is now beginning to emerge that miRNAs, short, non-coding RNAs, which post-transcriptionally regulate gene expression, play a key role in the regulation of innate and adaptive immunity in humans and mice. Little is currently known, however, regarding the importance miRNAs in regulating the host response to infection in agriculturally important animals, such as cattle. Mastitis is an inflammatory disease of the mammary gland caused either by infection or physical damage, which is associated with substantial economic losses. In this study, we report a next generation sequencing approach to profile the expression of bovine miRNAs in primary bovine mammary epithelial (BMEs) cells challenged with a bovine mastitis pathogen, Streptococcus uberis (0140J). Computational analysis has been undertaken on 450 million raw sequence reads and revealed that 20% of known bovine miRNAs are expressed in BMEs. Furthermore, 22 miRNAs were found to be differentially expressed over the 6 hour time-course. We have completed miRNA target prediction analysis and found that the target genes of down-regulated miRNAs are enriched for having a role in innate immunity, and pathways associated with target genes of up-regulated miRNA correspond to previously reported mastitis-relevant pathways. In addition, we report 21 potentially novel bovine miRNAs that have not previously been described, two of which have close human orthologs. This study provides new insight into the regulation of miRNAs in the host response to infection at an unprecedented level in any species.

overall_design: 24 miRNAseq libraries were prepared from 3 infected and 3 control replicates at 1, 2, 4 and 6 hours.

first: Amir

last: Foroushani

first: Kieran

last: Meade

first: Matthew

last: McCabe

first: Cliona

last: O’Farrelly

first: David

last: Lynn


title: Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells [ChIP-Seq]

overall_design: Native ChIP analysis of three histone post-translational modifications (H3K4me3, H3K27me3, H3K27ac) in two mouse embryonic stem cell (ESC) lines (control and H3.3-depleted). Inputs sequenced as control. Native ChIP analysis of H3.3B-HA in control and Suz12-/- ESCs. Crosslinking ChIP analysis of histone H3 using a general H3 antibody in two ESC lines (control and H3.3-depleted). Crosslinking ChIP analysis Hira, UTX, and Jmjd3 in wild type and H3.3 KO ESCs.


title: Flexible chromatin loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire

city: Vienna

first: Meinrad

last: Busslinger

address: Dr. Bohrgasse 7

country: Austria

institute: Instutute of Molecular Pathology

summary: The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH- rearranged gene segment in the proximal Igh domain. By high-resolution mapping of long-range interactions, we now demonstrate that an array of local interaction domains establishes the three- dimensional structure of the extended Igh locus in lymphoid progenitors and thymocytes. In pro- B cells, these local domains engage in long-range interactions across the entire Igh locus, which depend on the transcription factors Pax5, YY1 and CTCF. The large VH gene cluster thereby undergoes flexible long-range interactions with the more rigidly structured 3’ proximal domain, which ensures that all VH genes can participate with similar probability in VH-DJH recombination to generate a diverse antibody repertoire. Notably, these long-range interactions appear to be an intrinsic feature of the VH gene cluster, as they are still generated upon mutation of the Eμ enhancer, IGCR1 insulator or 3’ regulatory region present in the 3’ proximal Igh domain.

overall_design:
4C sequencing from mutliple celltypes with multiple viewpoints; uneven number of replicates
ChIP-Seq

first: Jasna

last: Medvedovic

first: Anja

last: Ebert

first: Markus

last: Jaritz

first: Hiromi

last: Tagoh


title: Genomewide analysis of U1C-dependent alternative splicing

city: Giessen

first: Lee-Hsueh

last: Hung

state: Hessen

address: Heinrich-Buff-Ring 58

department: Biochemistry

institute: University of Giessen

summary: To investigate whether U1C plays a role in splicing regulation in human system, we performed siRNA-mediated knockdown of U1C in HeLa cells and analyzed alternative splicing patterns by high-throughput RNA sequencing (RNAseq)

overall_design: RNAseq performed with poly(A)+ selected total RNA from U1C-knockdown and control-treated HeLa cells

first: Albrecht

last: Bindereif


title: Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells

first: Laura

last: Banaszynski


title: Genome-wide analysis of BRCA1 and PALB2 [ChIP-seq and RNA-seq]

first: Alessandro

last: Gardini

summary: Breast and ovarian cancer susceptibility genes, BRCA1 and PALB2 have enigmatic roles in cellular growth and mammalian development.  While these genes are essential for growth during early developmental programs, inactivation later in adulthood leads to increased growth and formation of tumors, leading to their designation as tumor suppressors.  We performed genome-wide analysis assessing their chromatin residence and gene expression responsiveness using high throughput sequencing in breast epithelial cells.  These experiments revealed a critical role for BRCA1 and PALB2 in transcriptional responsiveness to NF-kB, a crucial mediator of growth and inflammatory response during development and cancer.  Importantly, we also uncovered a vital role for these proteins in response to retinoic acid (RA), a growth inhibitory signal in breast cancer cells, which may constitute the basis for their tumor suppressor activity.

overall_design: Comparison of the genome wide profiles of the BRCA protein complex (BRCA1 and PALB2) and phosphorylated RNAPII (P-Ser2) in MCF10A cells by ChIP-seq. Effect of BRCA1 and PALB2 knockdown (shRNAs)  on transcription was  assessed by RNA-seq.

first: Matteo

last: Cesaroni

first: Ramin

last: Shiekhattar


title: Transcriptional Characterization of Streptococcus pneumoniae Interacting with Human Pharyngeal Cells

city: Baltimore

first: Jerry

last: Liu

address: 801 W. Baltimore St.

institute: IGS

summary: We used a combination of adherence assays, mutagenesis and functional genomics to identify novel factors involved in adherence.  This work identifies a list of novel potential pneumococcal adherence determinants.

overall_design: S. pneumoniae TIGR4 transcriptome was sequenced using RNA-Seq on the Illumina GA.  High-quality, single ended, 36bp reads were mapped to the TIGR4 genome.

first: Sheila

middle: Z

last: Kimaro-Mlacha

first: Sandra

last: Romero-Steiner

first: Julie

middle: C

last: Dunning-Hotopp

first: Nikhil

last: Kumar

first: Nadeeza

last: Ishmael

last: Riley

first: Umar

last: Farooq

first: Todd

last: Creasy

first: Luke

last: Tallon

first: Xinyue

first: Erin

last: Hine

first: Cynthia

middle: S

last: Goldsmith

first: Jacquelyn

last: Sampson

first: George

last: Carlone

first: Susan

last: Hollingshead

first: J

last: Scott

first: Herve

last: Tettelin


title: miRNA sequencing of 10 pairs samples between kidney normal tissues and cancer tissue

status: Public on Sep 29 2013

city: shenzhen

first: JACK

last: LI

address: beishangongyequ

institute: beijing genomics insititute

summary: we combined their genome-wide profiles of tumors and normal adjacent tissues in 10 ccRCC patients. The results showed that 283 miRNAs were down-regulated and 187 up-regulated, meanwhile 7473 mRNAs were up-regulated and 3439 down-regulated in ccRCC. Expressions of 12 miRNAs and genes were validated in 10 patients we studied by RT-qPCR (validation rate from 60% to 100%). Differentially expressed gene analysis showed the collective change of miR-200 and SLC22A gene family, and pathway analysis revealed down-regulation of multiple metabolic pathways and up-regulation of focal adhesion, ECM-receptor interaction, etc. Moreover, A miRNA cluster located in chromosome Xq27.3 were significantly down-regulated in ccRCC, which were verified in 53 ccRCC patients by RT-qPCR (validation rate from 63.8% to 88.6%).56 and 586 potentially novel miRNAs and mRNA we detected according to statistical analyses. In addition, 14 miRNA candidates were randomly selected to validate using qRT-PCR and Sanger sequcencing technology, 10 of out 14 could be successfully amplified,.Plus, the sequences of all 5 randomly selected amplified products were confirmed by cloned Sanger sequencing.Our data demonstrated a combined profiling of miRNAs and mRNAs in ccRCC, which showed the decreased metabolism and the perturbation of renal cell function. Moreover, this study identified the expression alteration of a miRNA cluster on Xq27.3, which suggested its potential function in carcinogenesis of ccRCC

overall_design: Examination of 10 pairs of kidney normal cells and cancer cells .

first: Jiahao

last: Chen

first: Zhizhong

last: Li


title: TALEN-based knockout of mir-141 and mir-200c in SK-BR-3 cells

status: Public on Sep 27 2013

city: Hwasun, Jeollanam-do

first: Young-Kook

last: Kim

address: 264, Seoyang-ro

department: Department of Biochemistry

country: South Korea

institute: Chonnam National University Medical School

summary: We used transcription activator-like effector nucleases (TALENs) to generate knockout cells for two related microRNAs (miRNAs), mir-141 and mir-200c, which belong to the deeply conserved mir-200 family. By carrying out deep sequencing, we identified the target genes of each miRNA. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppressed largely non-overlapping groups of targets.

overall_design: Analysis of global mRNA level change in mir-141 and mir-200c knockout compared to wild type cells

first: Narry


title: Next-Generation Sequencing for Small RNA Application in Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia

first: Mien-Chie

last: Hung

summary:
Purpose: The goal of this study is to identify the miRNA clusters that are regulated by EGFR under normoxia or hypoxia.
Method: Total RNAs were extracted from HeLa cells expressing scrambled control or EGFR shRNA-E1 that cultured under normoxia or hypoxia (1% O2) for 24h. Customized Next-Generation RNA deep sequencing, including both small RNA application and whole transcriptome analysis, was performed according to the standard procedure instructed by Applied Biosystems. For small RNA analysis, library inserts were size selected between 18 and 40nts and analyzed using CLC Genomics Workbench 4.7.1. 35nt colorspace reads were trimmed of adaptor sequence and mapped against human pre-miR sequences (miRBase version 16.0). Values of reads per million mapped reads (RPM) were based on mapped reads with no more than 2 mismatches total. A read was considered to come from a mature miRNA if it mapped to pre-miRNA sequences with no more than three upstream or downstream bases, and missing no more than two upstream or downstream bases from predicted mature or mature* sequences as defined in miRBase version 16.0. All the other pre-miRNA mapped reads were assigned as pre-miRNA signal. qRT–PCR validation was performed using TaqMan and SYBR Green assays.
Results: Deep sequencing analysis identified specific miRNA clusters that their maturation (miRNA processing efficacy was reflected by the relative expression of precursor miRNAs affected by EGFR compared with the relative expression of mature miRNAs affected by EGFR) were regulated by EGFR under normixa or specifically in response to hypoxia. Top miRNA candidates that regulated by EGFR in response to hypoxia were further verified by TaqMan and SYBR Green qRT-PCR assays.
Conclusion: Next-Generation Deep Sequencing for small RNA analysis revealed a novel function of EGFR involved in miRNA maturation in response to hypoxic stress.

overall_design: RNA profiles of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) that cultured under normoxia or hypoxia (1% O2) for 24h were generated by AB SOLiD next-generation sequencing. S: HeLa expressing scrambled control cultured under normoxia; A1: HeLa expressing EGFR shRNA-E1 cultured under normoxia; HS: HeLa expressing scrambled control cultured under hypoxia for 24h; HA1: HeLa expressing EGFR shRNA-E1 cultured under hypoxia for 24h. In total, 4 biological samples with no replicates resulted in 4 small RNA profiles.

first: Jia

last: Shen

first: Brian

last: James

first: Xiuping

last: Liu

first: Chang-Gong


title: Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3’ processing

city: Irvine

first: Yongsheng

last: Shi

address: University of California, Irvine B240, Med Sci I

institute: University of California, Irvine

summary: Our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3’ processing factors and provided new insights into the regulatory mechanisms of mRNA 3’ processing.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: CstF64tau iCLIP-seq in HeLa cells;  direct RNA sequencing (DRS) of control HeLa cells, CstF64tau-RNAi cells and SELEX-seq for RNA control pool, CstF64 and CstF64tau.

first: Chengguo

last: Yao

first: Eun-A

last: Choi

first: Lingjie

last: Weng

first: Xiaohui

last: Xie

first: Ji

last: Wan

first: Yi

last: Xing

first: James

first: Patricia

first: John

last: R.Yates III


title: Fungal-Specific Transcription Factor AbPf2 Activates Pathogenicity in Alternaria brassicicola

city: Honolulu

first: Yangrae

last: Cho

state: HI

address: 3190 Maile Way, St John 317

department: Plant and Environmental Protection Sciences

institute: University of Hawaii at Manoa

summary: Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen.  Molecular determinants of its life style shift between saprophyte and pathogen, however, are unknown.  To identify these determinants we studied nonpathogenic mutants of a transcription factor-coding gene, AbPf2.  Frequency and timing of germination and appressorium formation on host plants were similar between the nonpathogenic Δabpf2 mutants and wild-type A. brassicicola.  The mutants were also similar in vitro to wild-type A. brassicicola in vegetative growth, conidium production, and responses to chemical stressors, such as a phytoalexin, reactive oxygen species, and osmolites.  The mutants, however, did not penetrate host plant tissues, though their hyphae continued to grow on the plant surface.  Transcripts of the AbPf2 gene increased exponentially soon after wild-type conidia encountered their host plants.  A small amount of AbPf2 protein, monitored by fused green fluorescent protein, was located in both the cytoplasm and nuclei of young, mature conidia.  The protein level decreased during saprophytic growth but increased several-fold during pathogenesis.  Levels of both the proteins and transcripts sharply declined following colonization of host tissues beyond the initial infection site.  When the transcription factor was expressed at an induced level in the wild type during early pathogenesis, the expression of 106 fungal genes was down-regulated in the Δabpf2 mutants.  Notably, 33 of the 106 genes encoded secreted proteins, including eight putative effector proteins.  Plants inoculated with Δabpf2 mutants expressed higher levels of genes associated with photosynthesis, the pentose phosphate pathway, and primary metabolism, but lower levels of defense-related genes.  Our results suggest that conidia of A. brassicicola are programmed as saprophytes, but become parasites upon contact with their hosts.  AbPf2 coordinates this transformation by expressing pathogenesis-associated genes, including those coding for effectors.

overall_design:
Twenty seven Ar. thaliana (Col-0) were spray-inoculated until run-off with 5x105 conidia per milliliter of Δabpf2-5 mutant or the wild type.  Tissues containing both host plant tissue and fungal hyphae were harvested at 12 hours postinoculation and 48 hours postinoculation from three plants for each sample and immediately frozen in liquid nitrogen to fix gene expression profiles.

The frozen tissues were ground and total RNA was extracted using an RNeasy kit.  Residual DNA was digested in columns with RNase-free DNase following the manufacturer’s protocol (Qiagen, Palo Alto, CA). Three biological replicates were prepared for the mutant and three for the wild type and their gene expression profiles compared.  We constructed strand-specific sequencing libraries using the TruSeq™ RNA Sample Prep Kit (Illumina, San Diego, CA) from total RNA following the manufacturer’s protocol.  Each sample RNA was used to construct a library with a unique index primer.  A total of six index primers were used to construct six libraries.  All six libraries were mixed and determined 100 nucleotide-long sequence tags using Illumina Hiseq2000 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.  Image analysis, base-calling, and quality checks were performed with the Illumina data analysis pipeline CASAVA v1.8.0.


title: Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells [CATCH-IT]

first: Laura

last: Banaszynski

overall_design: CATCH-IT analysis of five embryonic stem cell lines (control, H3.3 KD1, and H3.3 KD2; wild type and Hira-/-)

first: Scott

last: Dewell

first: Deyou

last: Zheng

first: C

middle: D

last: Allis


title: Genome-wide mapping of open chromatin in mouse embryonic stem cell

city: Shenzhen

first: Xia

last: Li

address: No.1 Fuhua Road, Futian District, Shenzhen, P.R. China

department: Department of Pathology

institute: Shenzhen Traditional Chinese Medicine Hospital

summary: Micrococcal nuclease was used to digest mESC nuclei under a mild digestion condition to release the relatively open chromatin, and under an extensive digestion condition to release the genome chromatin. By comparing the genome distribution of mono-nucleasome under these two conditions, open chromatin regions were detected.

overall_design: One mild digestion and one extensive digestion was applied to the mESC cells, followed by sequencing.


title: Comparison of root transcriptomes and expression of genes involved in main medicinal secondary metabolites from Bupleurum chinense and B. scorzonerifolium, the two Chinese official Radix bupleuri source species

city: Beijing

first: Sui

last: Chun

state: Beijing

address: No. 151,Malianwa North Road, Hai Dian District

institute: Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences & Peking Union Medical College

summary: Radix bupleuri is a traditional Chinese crude drug and it is still widely used in contemporary medicine. Bupleurum chinense DC. and B. scorzonerifolium Willd. are officially recorded as the source species of Radix bupleuri in Pharmacopoeia of the People’s Republic of China. The obvious difference in chemical active components and pharmacological action of Radix bupleuri derived from the two species has caused attention. But to date, no definite criterions and regulates are issued based on plant species sources to direct the standard uses of Radix bupleuri for their extracts and traditional decoction pieces in medical market. Using 454-FLX pyrosequencing with one half-plate run for each cDNA library, a total of 419,576 and 416,112 reads, among those 313,483 and 342,263 high quality ESTs were obtained for B. chinense and B. scorzonerifolium, respectively. The average read length was 279 bp and 310 bp, respectively. The assembled 28,897 and 30,912 unigenes for B. chinense and B. scorzonerifolium were successively annotated based on sequence similarity by major public nucleotide/protein databases, Nt, Nr, SwissProt, KEGG, COG, Interpro and GO. Based on the alignments, 71.9% (20,787) and 75.8% (23,426) of the total unigenes for B. chinense and B. scorzonerifolium could be matched to nucleotides from NCBI Nt database. Certain percent from 27.1% (7,820) to 70.8% (20,461) of the total unigenes for B. chinense, and from 26.4% (8,145) to 72.1% (22,289) of the total unigenes for B. scorzonerifolium, could be matched to proteins from different databases. For the same database, similar percent of the total unigenes for B. chinense and B. scorzonerifolium were annotated. A total of 17,117 (59.2%) and 19,416 (62.8%) unigenes for B. chinense and B. scorzonerifolium could be found homologous genes in opposite dataset. According to KEGG annotation, 3,637 and 3,821 unigenes were assigned relating to metabolism in datasets of B. chinense and B. scorzonerifolium, respectively. Carbohydrate metabolism, energy metabolism and amino acid metabolism were the highest three groups in metabolism category and followed by lipid metabolism group for B. chinense. In comparison, lipid metabolism group had most unigenes for B. scorzonerifolium and it had much more unigenes than the same group for B. chinense (827 to 562). A total of 70 and 52 unigenes were related to metabolism of terpenoids and polyketides in two separate datasets, which seems agree with the situation of higher content of terpenoid saikosaponins in B. chinense than B. scorzonerifolium in some extent. To compare the biosynthesis of main bioactive components in roots of B. chinense and B. scorzonerifolium, putative genes which may participate in the biosynthesis of terpenoid, triterpenoid, sterol, ligan and flavonoid were searched according to unigenes annotation. Most lignan and flavonoid biosynthesis related genes were found in both datasets of B. chinense and B. scorzonerifolium, while some terpenoid or triterpenoid related genes were dominantly found in the dataset of B. chinense. This reflected in some extent that these genes expressed in low levels and relative metabolites were biosynthesized in lower quantities in roots of B. chinense than those of B. scorzonerifolium. Besides abovementioned comparison on two transcriptome, the expression of some genes involved in biosyntheses of main medicinal secondary metabolites were analyzed by qRT-PCR in roots of the two species. These molecular biological data will make a useful clue and arouse more studies on the reasonable application of the two Bupleurum species.

sample_organism:
Bupleurum scorzonerifolium
Bupleurum chinense

overall_design: The fine roots of two-year old plants of B. chinense and B. scorzonerifolium planted in the same place at the same time were collected at the usual harvest time of medicinal materials of Bupleurum. Those roots from the two species plants were used for 454 sequencing.

first: Chun

last: Sui

first: Jianhe

last: Wei


title: Miz1 is required to maintain autophagic flux

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary: Miz1 is a zinc finger protein that regulates expression of cell cycle inhibitors as part of a complex with Myc. Cell cycle-independent functions of Miz1 are poorly understood. Here, we use a Nestin-Cre transgene to delete an essential domain of Miz1 in the central nervous system (Miz1ΔPOZNes). Miz1ΔPOZNes mice display cerebellar neurodegeneration characterized by the progressive loss of Purkinje cells. Chromatin immunoprecipitation sequencing and biochemical analyses show that Miz1 activates transcription upon binding to a non-palindromic sequence present in core promoters. Target genes of Miz1 encode regulators of autophagy and proteins involved in vesicular transport that are required for autophagy. Miz1ΔPOZ neuronal progenitors and fibroblasts show reduced autophagic flux. Consistently, polyubiquitinated proteins and p62/Sqtm1 accumulate in the cerebella of Miz1ΔPOZNes mice, characteristic features of defective autophagy. Our data suggest that Miz1 may link cell growth and ribosome biogenesis to the transcriptional regulation of vesicular transport and autophagy.

overall_design: ChIP-Seq with H190 and G18 on an Illumina Genome Analyzer IIx.

first: Elmar

last: Wolf

first: Anneli

last: Gebhardt

first: Daisuke

last: Kawauchi

first: Susanne

last: Walz

first: Björn

last: von Eyss

first: Nicole

last: Wagner

first: Christoph

last: Renninger

first: Georg

last: Krohne

first: Esther

last: Asan

first: Martine

last: Roussel


title: Expression profiling of genes involved in somatic and zygotic embryo development in cotton

city: Wuhan

first: Fangyan

last: Jin

state: Hubei

address: No.1, Shizishan Street

institute: Huazhong Agriculture University

summary: We used a next-generation sequencing approach, Illumina Digital Gene Expression II (DGEII) technology, to Genome-wide profiling of gene expression during somatic (SE) and zygotic embryo (ZE) development. As a result, 4242 differentially expressed genes (DEGs) were identified between SE and ZE. Expression pattern cluster and functional classification analysis of the differentially expressed genes revealed that large number of genes indicative of response to stress highly expressed in somatic embryos. Combined with KEGG analysis and BLAST annotation, genes related to stress response in DEGs have been comprehensively analyzed, mainly including: ABA biosynthesis, ABA and JA (jasmonic acid) response, LEA (late embryogenesis abundant protein) family members, RD (responsive to dehydration) and ERD (early responsive to dehydration) family members, Hot Shock Proteins, WRKY family members and NAC family members. Semi-quantitative Reverse Transcriptase-PCR (RT-PCR) analysis was performed on 27 selected genes and and the results enhance that genes related to stress response were involved in SE development. Our main goal is to adapt the RNA-Seq technology to this notable development process and to analyse the gene expression profile. This is a systematical expression analysis focusing on somatic embryo development, and also a global comparison between somatic and zygotic embryo at transcript level. Therefore, this study might bring new insights into the regulation of somatic embryo development.

overall_design: An analysis of differentially expressed genes at 3 parallel stages during somatic and zygotic embryo development in cotton by next-generation sequencing, using Illumina Digital Gene Expression II (DGE II) .

first: Xianlong

last: Zhang


title: Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [MeDIP-seq]

city: Torino

first: Francesco

last: Neri

state: Italy

address: Via Nizza 52

country: Italy

institute: University of Torino

summary: The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methylase that has been previously shown to cooperate with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESC) but its function in these cells is unknown. We here report that Dnmt3L is required for the differentiation of ESC into primordial germ cells (PGC) through activation of the homeotic gene Rhox5. By genome-wide analysis we found that Dnmt3L is a positive regulator of methylation at gene bodies of housekeeping genes and a negative regulator of methylation at promoters of bivalent genes. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyl transferases Dnmt3a and Dnmt3b to maintain low the methylation level at H3H27me3 regions. Thus in ESC, Dnmt3L counteracts the activity of de novo DNA methylases to keep low the level of DNA methylation at developmental gene promoters.

overall_design: Examination of 5mC in shGFP and shDnmt3L ESC by MeDIP-Seq

first: Salvatore

last: Oliviero


title: Whole genome bisulfite sequencing of rdm16ros1, ros1, nrpd1ros1 mutants and C24 wild-type

city: West Lafayette

first: Jian-Kang

last: Zhu

state: IN

address: 625 Agriculture Mall Dr.

department: Department of Horticulture and Landscape Architecture

institute: Purdue University

summary: Using whole genome bisulfite sequencing to provide single-base resolution of DNA methylation status in rdm16ros1, ros1, nrpd1ros1 mutants and examine the effect of RDM16 on DNA methylation

overall_design: 4 samples examined: C24 wild type with RD29A-LUC transgene, rdm16ros1 double mutant, ros1 mutant, nrpd1ros1 mutant (all in C24 background with RD29A-LUC transgene)

first: Chao-Feng

last: Huang

last: Miki

first: Kai

last: Tang

first: Zeyang

last: Ma

first: Zhimin

last: Zheng

first: Wei

last: Chen

first: Lan

last: Yang

first: Heng

first: Renyi

last: Liu

first: Xinjian

last: He


title: Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [ChIP-seq]

summary: The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methylase that has been previously shown to cooperate with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESC) but its function in these cells is unknown. We here report that Dnmt3L is required for the differentiation of ESC into primordial germ cells (PGC) through activation of the homeotic gene Rhox5. By genome-wide analysis we found that Dnmt3L is a positive regulator of methylation at gene bodies of housekeeping genes and a negative regulator of methylation at promoters of bivalent genes. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low the methylation level at H3H27me3 regions. Thus, in ESC, Dnmt3L counteracts the activity of de novo DNA methylases to keep low the level of DNA methylation at developmental gene promoters.

overall_design: Examination of Ezh2 ChIP-Seq in shGFP and shDnmt3L ESC. Examination of Dnmt3L ChIP-Seq in shGFP and shSuz12 ESC.


title: Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma

first: JACK

last: LI

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

overall_design: Refer to individual Series


title: Genome-wide profiling of Argonaute-chromosome interactions [ChIP-Seq]

city: San Francisco

first: Vera

address: 1450 Third Street, Helen Diller Cancer Research Building, Room 302

department: Urology

institute: UCSF

summary: Argonuate proteins are the central components of the RNA-induced silencing complex (RISC) which execute small RNA-mediated gene regulation mainly in the cytoplasm and their nuclear functions in mammalian cells remain largely unknown.  Here we show that in human cancer cells Ago1 is pervasively associated with chromosomal loci throughout the genome with the association centered mostly on actively transcribed promoters. Ago1 but not Ago2 physically interacts with RNA polymerase II (RNAP II) in the nucleus. Moreover, genes bound by Ago1 are significantly enriched for several cancer related pathways. Detailed analysis of these genes including PIK3CA, PRKCH, CDC6 and RRM1 verified Ago1-mediated transcriptional gene regulation through Ago1-promoter interactions. We also found a strong correlation between Ago1-bound genes and genes downregulated by Ago1 depletion which, phenotypically, leads to inhibition of proliferation in different cancer cells.  Therefore, our findings reveal the first landscape of human Ago1-chromosomal interactions which may drive oncogenic programs in cancer cells

overall_design: Examination of Ago-DNA interactions in human cancer cells

first: Jiashun

first: Long-Cheng

last: Li

first: Hao


title: Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells

first: Francesco


title: Characterization of the genetic mouse model of non-small cells lung cancer p53R172H∆g/KrasG12D

status: Public on Sep 25 2013

first: Raffaele

last: Calogero

state: To

department: Molecular Biotechnology Center

summary:
Background: Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a great need for additional effective therapies. In this work we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC cancer, as biological reservoir for potential therapeutic targets and biomarkers.
Results:RNA-seq profiling was performed on total RNA extracted from lungs of 30 week-old p53R172H∆g/KrasG12D and wild type mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in p53R172H∆g/KrasG12D tumors. Differential expression at exon-level detected 33 genes with differential exon usage.
The study provides a complete transcription overview of the p53R172H∆g/KrasG12D mouse NSCLC model

overall_design: Lung mRNA profiles of 30-week old wild type (WT) and p53R172H∆g/KrasG12D mice were generated by deep sequencing, in duplicate using Illumina HiSeq2000.

first: Federica

last: Riccardo

first: Maddalena

last: Arigoni

first: Genny

last: Buson

first: Elisa

last: Zago

first: Manuela

last: Iezzi

first: Dario

last: Longo

first: Matteo

last: Carrara

first: Alessandra

last: Fiore

first: Simona

last: Nuzzo

first: Silvio

last: Bicciato

first: Patrizia

last: Nanni

first: Lorena

last: Landuzzi

last: Cavallo

last: Clogero

first: Elena

last: Quaglino


title: Genome-wide maps of XBP1 binding sites in different breast cancer cell lines.

status: Public on Sep 26 2013

city: Boston

first: Xiaole

middle: Shirley

last: Liu

state: MA

address: 450 Brookline Avenue, CLS 11007

department: Department of Biostatistics and Computational Biology

institute: Dana-Farber Cancer Institute, Harvard School of Public Health


title: Genome-wide maps of XBP1 binding sites in different breast cancer cell lines [ChIP-Seq]

summary: We report the application of ChIP-seq, which combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing, to map genome-wide XBP1 binding sites in different breast cancer cell lines. We showed that HIF1α motif was enriched in XBP1 binding sites in triple negative breast cancer (TNBC) cell lines, but not enriched in ER positive breast cancer cell line. We also demonstrated that different breast cancer cell lines of the same sub-type had similar XBP1 binding sites, whereas different breast cancer sub-types had majorly different XBP1 binding sites. Finally, a model was applied to integrate XBP1 ChIP-seq data with expression data to predict XBP1's direct targets in TNBC cell line; the predicted direct targets were shown to be predictive of patient survival, and the prediction power was specific to TNBC patients. The above evidence indicates that XBP1 performs important functions in TNBC by interacting with HIF1α, and such regulation mechanism is specific to TNBC, which is later proved by follow-up experiments.This study represents the first detailed anaysis of XBP1 binding sites in different breast cancer cell lines.

overall_design: Examination of XBP1 binding sites in 2 cell types (3 cell lines).

first: Chen

last: Xi

first: Glimcher

last: Laurie


title: Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites [GP5d/siRAD21 ChIP-Seq experiments]

status: Public on Sep 28 2013

city: Stockholm

first: Martin

last: Enge

address: CCK, Z4

department: Dep of Oncology-Pathology

country: Sweden

institute: Karolinska Institute

summary: During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation.

overall_design: Examination of TF binding by ChIP-seq in a CRC cell-line treated with non-targeting siRNA, or siRNA specific to the cohesin subunit RAD21.

first: Jian

last: Yan


title: The Aurora B kinase and the polycomb protein Ring1B combine to regulate active promoters in quiescent lymphocytes

city: London

first: Gopuraja

last: Dharmalingam

address: Faculty of Medicine, Imperial College, Hammersmith Hospital Campus

department: Epigenetics section

institute: MRC London Institute of Medical Sciences

first: Niall

last: Dillon

first: Alberto

last: Frangini