title: Deterministic direct reprogramming of somatic cells to pluripotency [ChIP-Seq]

city: Rehovot

first: Asaf

last: Zviran

address: 234 Herzl Street

department: Molecular Genetics

institute: Weizmann Institute of Science

summary: Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution.

overall_design: Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors). Three histone modifications are given: H3K4me3, H3K27me3 and H3K27ac. In addition, binding data of Mbd3, Mi2B and Oct4 is given in various stages. Please note that 20 independent clonal series generated in our study were carrying either the GOF18 deltaPE-Oct4-GFP transgenic reporter (Addgene plasmid 52382) or the complete GOF18 Oct4-GFP transgenic reporter (Addgene plasmid 60527), the clonal series selected for genomic analysis included an Mbd3+/+ clone that carries the GOF18 deltaPE-Oct4-GFP transgenic reporter, andMbd3flox/-and Mbd3-/- cells that carry the GOF18 Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements). These reporters can be identified when analyzing the Oct4 locus in genomic DNA input datasets. As we do not use Oct4–GFP or any other selection for sorting cells before conducting genomic experiments, the difference in transgene reporters would not influence the interpretation of our genomic analysis data in any way. In these genomics studies, the endogenous Nanog and Oct4 loci are not manipulated and are identical between all cell lines because the Oct4–GFP reporters were introduced via random transgenesis and validated for specificity. Notable, for the mouse induced pluripotent stem (iPS) cell efficiency results presented in our study (for example, in Figs 1 and 2, and Supplementary Videos 1–4), cell lines carried the matched Nanog–GFP knock-in reporter or Oct4– GFP transgenic reporter (containing both distal and proximal enhancer elements as delineated in Extended Data Fig. 3a). Thus, all iPS cell efficiency and kinetic comparisons were conducted by using matched and validated pluripotency reporter systems.

first: Yoach

last: Rais


title: Transcriptional profiles by deep sequencing (RNA-seq) of in vivo-generated mouse iPSCs, in vitro-generated mouse iPSCs, and mouse ESCs

status: Public on Sep 12 2013

city: Barcelona

first: Manuel

last: Serrano

state: Barcelona

address: Parc Científic de Barcelona, C/ Baldiri Reixac 10

department: Molecular Medicine

country: Spain

institute: IRB

type: Expression profiling by high throughput sequencing

summary: We have generated “reprogrammable” transgenic mice that ubiquitously express the four Yamanaka factors in an inducible manner. Transitory induction of the transgene results in multiple teratomas emerging from a variety of organs, thus indicating that full reprogramming into iPSCs can occur in vivo. By performing bone marrow transplant experiments, we demonstrate that both hematopoietic cells, as well as non-hematopoietic cells can be reprogrammed in vivo. Remarkably, reprogrammable mice also present circulating iPSCs in the bloodstream (in vivo-iPSCs) with all the expected properties of bona fide iPSCs. Moreover, in contrast to in vitro-iPSCs or embryonic stem cells (ESCs), in vivo-iPSCs have an increased capacity to undergo trophectoderm lineage differentiation, which suggests that in vivo-iPSCs are more plastic or primitive than in vitro-generated iPSCs or ESCs.

overall_design: 6 clones of in vivo-generated iPSCs, 5 indendent clones of in vitro-generated iPSCs, and 3 clones of established ESCs

first: Maria

last: Abad

first: Osvaldo

last: Graña


title: The Small RNA Profile of Silkworm Hemolymph Using Deep Sequencing

city: Chongqing

first: Ling

last: Jia

address: Tiansheng Road 1

country: China

institute: State key laboratory of silkworm genome biology

summary: Human utilization of the mulberry-silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species (Morus notabilis C. K. Schneider). In the 330 Mb genome assembly of M. notabilis, we identified 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which were supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating its spread to Europe, Africa, and America. It is among few eudicots but several Rosales not preserving genome duplications in more than 100 million years – however neopolyploid series in mulberry and several others suggest that new duplications may confer benefits. Strikingly, five predicted mulberry miRNAs were found in the hemolymph and silkglands of silkworm, suggesting profound molecular level interactions that promise to expand knowledge of plant-herbivore relationship which constitute key elements of most terrestrial habitats. In addition, we investigated the characters of hemolymph small RNA.

overall_design: small mRNA profiles of silkworm hemolymph in the fifth instar day-5 silkworm  were generated by deep sequencing, in twice, using Illumina Hiseq 2000.

first: Ningjia

last: He

first: Xiwu

last: Qi


title: Long-range chromatin contacts reveal a role for the pluripotency and Polycomb networks in genome organization

city: Los Angeles

first: Kathrin

last: Plath

address: 615 Charles E. Young Drive South

country: USA

institute: UCLA

summary: We mapped long-range chromatin interactions in embryonic stem cells (ESCs), iPSCs, and fibroblasts, and uncovered an ESC-specific genome organization that is gradually re-established during reprogramming. Confirming previous results, we show that open, accessible and closed chromatin character is the primary determinant of long-range chromatin interaction preferences. Importantly, we find that in ESCs, genomic regions extensively occupied by the pluripotency factors Oct4, Sox2, and Nanog preferentially co-localize. Similarly, regions strongly enriched for Polycomb-proteins and H3K27me3 frequently interact, and loss of the Polycomb-protein Eed diminishes these interactions without dramatically changing overall chromosome-conformation. Consistent with a spatial segregation of transcriptional networks in ESCs, Nanog and Polycomb-proteins occupy distinct nuclear spaces. Together, our data reveal that transcriptional networks that govern ESC identity play a role in genome-organization.

overall_design: 4C-seq was performed using a variety of baits on range of mouse cell lines, namely, ESCs, reprogrammed iPSCs, partially reprogrammed pre-iPSCs, and differentiated MEFs. Additionally, 4C-seq was performed on a partially overlapping set of baits for a Eed mutant mouse ESC line and a sibling wild-type ESC line.

first: Matthew

last: Denholtz

first: Giancarl

last: Bonora

first: Constantinos

last: Chronis

first: Erik

last: Splinter

first: Wouter

last: deLaat

first: Jason

last: Ernst

first: Matteo

last: Pellegrini


title: Deterministic direct reprogramming of somatic cells to pluripotency

country: Israel

type: Genome binding/occupancy profiling by high throughput sequencing

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: chromatin reprogramming and differential gene expression in a model of EMT (spheroid A549 treated with TGF/TNF)

city: Charlottesville

first: Marcin

middle: Piotr

last: Cieslik

state: VA

address: 400 Stribling Ave Ext

institute: University of Virginia

first: Marty

middle: W

last: Mayo

first: Stefan

last: Bekiranov

first: Natalya

last: Baranova

first: David

last: Allison

first: Jake

last: Wamsley

first: Lisa

last: Gray

first: Sanjay

last: Chodaparambil

first: Manish

last: Kumar

first: Stephen

last: Hoang

first: Xiaojiang

last: Xu


title: Tgif1 regulates quiescence and self-renewal signaling pathways of hematopoietic stem cells (HSCs)

status: Public on Sep 10 2013

city: Nashville

first: Yan

last: Guo

state: TN

address: 639 Preston Research Bld

institute: Vanderbilt Cancer Center

summary: In this study we demonstrate that Tgif1 has a role in HSCs maintenance, self-renewal and quiescence. RNA sequencing data of LSK cells (HSCs enriched cell population) from Tgif1-/- and wild type mice  implicates that multiple pathways involved in HSC quiescence and self-renewal are disturbed in Tgif1 deficient mice.

overall_design: RNA expression profiles of  wild type (WT) and Tgif1-/- LSK cells were generated by RNA sequencing, in triplicate, using Illumina HiSeq 2000.

first: Ling

last: Yan

first: Rizwan

last: Hamid

last: Brandt

first: Yu

last: Shyr

last: Wotton

first: Bethany

last: Womack

first: Utpal

last: Dave

first: Chun

last: Li

first: Scott

last: Hiebert


title: Genome-wide measurement of Nkx-6.1 in mouse islets.

city: Philadelphia

first: Jonathan

last: Schug

state: Pennsylvania

address: 12-156 Smilow Ctr Trans Research

department: School of Medicine

institute: University of Pennsylvania

summary: We perfomed a ChIP-seq experiment to identify the location of binding sites for the transcription factor Nkx6-1 in mouse islets.

overall_design: A single set of islets were extracted from a mouse and Nkx-6.1 and input ChIP-Seq libraries were prepared and sequenced.

first: Brandon

last: Taylor

first: Maike

last: Sander


title: Active STAT5 in CD8 T cells

status: Public on Sep 13 2013

city: MARSEILLE

first: Nathalie

last: AUPHAN-ANEZIN

address: Parc Scientifique de Luminy

country: France

institute: CIML


title: Plants encode a general siRNA suppressor that is induced and suppressed by viruses

city: Castanet

first: Matthias

last: Zytnicki

address: 24, Chemin de Borde-Rouge

department: Applied Mathematics and Informatics

institute: INRA

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNAs play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode DICER-LIKE (DCL) RNaseIII enzymes, including DCL1, which produces miRNAs, and DCL2, DCL3 and DCL4, which produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Small RNA sequencing in plants over-expressing RTL1 or RTL2 or lacking RTL2 revealed that RTL1 over-expression inhibits the accumulation of all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. By contrast, RTL2 plays minor, if any, role in the small RNA repertoire. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by degrading dsRNA before they are processed by DCL2, DCL3 and DCL4. The substrate of RTL1 cleavage is likely long perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from short imperfect dsRNA. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. These results reveal another level of complexity in the evolutionary battle between viruses and plant defenses.

overall_design: Flower sRNA profiles in diverse conditions involving RTL1 and RTL2


title: Histone modification analysis in TGFbeta/TNFalpha treated A549 spheroid cultures

first: Marcin

last: Cieslik

summary: We studied the extent of chromatin remodeling in an in-vitro model of the epithelial-mesenchymal transition (EMT). EMT is induced in spheroid cultures (3D) using simultaneously two cytokines: TGFbeta and TNFalpha.

overall_design: The epithelial-mesenchymal transition (EMT) is a cellular de-differentiation process that has been implicated in cancer progression and metastasis. Increasing evidence suggests that EMT is regulated and established by epigenetic reprogramming, however a systems-level mechanism describing how chromatin remodeling contributes to the phenotypic switch is not known. We have generated genome-wide maps of 18 histone modifications/variants and variants in both the epithelial and mesenchymal states and quantified patterns of epigenetic changes at gene and enhancer loci. Clusters of these patterns reveal that EMT-related genes and their proximal enhancers are regulated through coordinated patterns of chromatin activation and repression at both gene and enhancer loci. At the cellular level, the remodeling of gene loci translates into a modular protein interaction network that recapitulates EMT-related signaling. Moreover, differentially activated or repressed enhancers are associated with two non-overlapping sets of transcription factors. We propose a chromatin-mediated regulatory feedback loop model where the NFkappaB and AP-1 transcription factors (TFs) bind activated enhancers, that regulate EMT-related genes, which in turn activate signaling pathways upstream of these TFs.

first: Marty

last: Mayo

first: Stefan

last: Bekiranov

first: Natalya

last: Baranova

first: David

last: Allison

first: Jake

last: Wamsley

first: Lisa

last: Gray

first: Sanjay

last: Chodaparambil

last: Kumar

last: Hoang


title: Deep sequencing reveals increased DNA methylation in chronic rat epilepsy (mRNA-seq)

status: Public on Sep 09 2013

city: Melbourne

first: Assam

last: El-Osta

address: 75 Commercial Road

country: Australia

institute: Baker Heart and Diabetes Institute

summary: Explore DNA methylation in chronic epilepsy and its relationship to gene expression.

overall_design: Examination of expression changes in pilocarpine-treated rats compared to controls and pilocarpine-treated rats on a ketogenic diet.

first: Katja

last: Kobow

first: Antony

last: Kaspi

first: K

middle: N

last: Harikrishnan

first: Katharina

last: Kiese

first: Mark

last: Ziemann

first: Ishant

last: Khurana

first: Ina

last: Fritzsche

first: Jan

last: Hauke

first: Eric

last: Hahnen

first: Roland

last: Coras

first: Angelika

last: Mühlebner

first: Ingmar

last: Blümcke


title: Deep sequencing reveals increased DNA methylation in chronic rat epilepsy


title: Deep sequencing reveals increased DNA methylation in chronic rat epilepsy (MBD-seq)

overall_design: Examination of methylation changes in pilocarpine-treated rats compared to controls and pilocarpine-treated rats on a ketogenic diet.


title: The expression analyses of trisomy 2L compared with normal

city: Columbia

first: Lin

last: Sun

state: MO

address: 311 Tucker Hall

department: Biological Sciences

institute: University of Missouri

summary: Dosage compensation was referred as an equalized X chromosome gene expression between males and females in Drosophila. And inverse dosage effects, produced by genomic imbalance, are believed to account for this modulated expression. Here we made a global expression comparison of trisomy 2L with on extra copy of chromosome 2 long arm to normal diploid with two copies of 2L with high throughput RNA-sequencing. We want to test how about the gene expression pattern changes in those comparisons, including the genes on varied chromosome 2 long arm, some other autosomal genes except chromosome 2L and X chromosome genes. Dosage compensation with an expression level similar to normal diploid  and inverse dosage effects should be detected.

overall_design:
Comapare the global expression of trisomy 2L samples with the normal diploids
Collected the females and males from trisomy 2L and Cantons and performed RNA-seq

first: James

middle: A

last: Birchler


title: Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression

city: Lyon

first: Mathilde

last: PARIS

address: 32-34 avenue Tony Garnier

department: Institut de Genomique Fonctionnelle de Lyon

institute: CNRS

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura
Drosophila virilis
Drosophila yakuba


title: RNA-Seq profiling unveils a non-canonical Wnt signalling signature in pancreas versus liver fate decision

status: Public on Sep 11 2013

city: London

first: Francesca

middle: M.

last: Spagnoli

address: Great Maze Pond

department: CGTRM

country: United Kingdom

institute: King's College London

summary: Understanding how distinct cell types arise from common multipotent progenitor cells is a major quest in stem cell biology. This knowledge will aid in the targeted differentiation and growth of stem cells, but also in the discovery of the basis of cellular plasticity and of how tissue programming can be controlled. The liver and pancreas share many aspects of their early development, being both specified in the same region of the endoderm, and, possibly, originating from a common progenitor. However, how pancreas versus liver cell fate decision occurs during embryogenesis and the molecular basis of this cellular plasticity are poorly understood. Here, we use RNA-Seq to define the molecular identity of liver and pancreas progenitors directly in mouse embryos and to investigate the mechanisms regulating the emergence of liver or pancreas as alternative fates from the endoderm. Progenitor cell-specific RNA was obtained from mouse Prox1-EGFP-labeled embryonic cells isolated by FACS at distinct developmental stages, before and after the onset of organogenesis. By integrating the temporal and spatial gene expression profiles, we found mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the non-canonical Wnt pathway as a potential developmental regulator of the pancreas versus liver fate decision, being expressed in the foregut endoderm, before the cell fate choice is made, and then maintained in pancreas progenitors but absent in hepatic progenitors. Moreover, when assayed in Xenopus embryos, the non-canonical Wnt pathway is able to promote pancreatic fate and repress hepatic fate in the endoderm, suggesting an ancient mechanism for controlling pancreas versus liver fate choice. We expect that this knowledge will be key to formulate reprogramming strategies to convert adult hepatic cells into pancreatic cells as a cell-based therapeutic approach for diabetes.

overall_design: We performed sequencing-based expression profiling (RNA-Seq) of hepatic and pancreatic progenitors in the mouse at two distinct developmental stages.

first: Nancy

last: Mah

middle: M


title: Role of RelA:CBP/p300 interaction on RelA-activated genes

city: San Diego

first: Harry

middle: Aaron

last: Birnbaum

state: California

address: 4406 Dawes Street

institute: UCSD Chem and Biochem

summary: A structure-function study of NF-kB subunit RelA and coactivator CBP/p300 interaction reveals the critical role of CBP/p300 in recruitment of RelA to its target promoter site.

overall_design: mRNA profiles of unstimulated or stimulated with TNFa rela-/- MEF reconstituted with RelA wild type or mutants were generated by deep sequencing, in duplicate

first: Sulakshana

middle: P

last: Mukherjee

first: Gourisankar

last: Ghosh

first: Alexander

last: Hoffmann


title: Lamin B1 depletion in senescent cells leads to large-scale changes in the chromatin landscape

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

first: Parisha

last: Shah

first: Greg

first: David

last: Nelson

first: Kajia

last: Cao

first: Varun

last: Aggarwala

first: Peter

middle: D

last: Adams

first: Shelley

middle: L

last: Berger


title: Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros (RNA-seq: sorted DP thymocytes)

status: Public on Sep 08 2013

first: Teresita

last: Arenzana

address: 675 Charles E. Young Dr. S., MRL 6-535

department: Microbiology, Immunology, and Molecular Genetics

summary: The C2H2 zinc finger is the most prevalent DNA-binding motif in the mammalian proteome, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros, a critical regulator of lymphopoiesis and leukemogenesis. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling (this GEO submission: RNA-Seg of whole thymus from wt and the two ZnF mutants), reveal that different subsets of fingers within multi-finger transcription factors can regulate distinct target genes and biological functions, and they demonstrate that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors.

overall_design: Ikaros RNA-Seq from double positive thymocytes comparing wt (n=2), Ikaros-ZnF1-/- mutant (n=2) and Ikaros-ZnF4-/- mutant (n=2)

first: Stephen

middle: T

last: Smale


title: Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros (RNA-seq: sorted proB cell Hardy Fractions B and C+C')

city: San Francisco

first: Hilde

last: Schjerven

state: CA

address: 513 Parnassus Ave

department: Laboratory Medicine

institute: UCSF

overall_design: RNA-Seq from sorted primary proB cell Hardy Fractions B and C+C', comparing wt, Ikaros-ZnF1-/- mutant and Ikaros-ZnF4-/- mutant.


title: Profiling microRNA expression in bovine alveolar macrophages using RNA-seq

city: Dunsany

last: Vegh

state: Meath

address: Grange, Batterjohn Crossroads

department: Animal & Bioscience Research Department

country: Ireland

institute: Teagasc

summary: In this study, we determined the miRNA expression profile of bovine alveolar macrophages, using next-generation sequencing strategy. On an Illumina HiSeq 2000 machine, we sequenced 8 miRNA libraries, prepared from small RNA fractions of alveolar macrophages isolated from 8 different healthy animals (Bos taurus). From the data, the potential novel miRNAs were predicted, and the expression levels of the known miRNAs were determined. We report that 80 known bovine miRNAs are expressed in bovine alveolar macrophages with >100 reads per million. The most highly expressed miRNA was bta-miR-21, followed by bta-miR-27a. Additionally, one putatively novel bovine miRNA was identified. To our knowledge, this is the first RNA-seq study to profile miRNA expression in bovine alveolar macrophages and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type.

overall_design: Examination of bovine alveolar macrophage miRNA profiles, using RNA-seq. Alveolar macrophages were isolated from lung lavages from 8 animals. Small RNA fractions were prepared from the cells using the Qiagen RNeasy Plus mini kit, and miRNA sequencing libraries were prepared using the Epicentre Scriptminer multiplex kit. The sequencing was performed on an Illumina HiSeq 2000 machine.

first: Matthew

middle: S

last: McCabe

first: Amir

middle: K

last: Foroushani

last: Magee

first: John

last: Browne

first: Nicolas

middle: C

last: Nalpas

first: Kevin

last: Conlon

middle: V

last: Gordon

first: Daniel

middle: G

last: Bradley

middle: E

last: MacHugh

middle: J

last: Lynn


title: Lamin B1 depletion in senescent cells triggers large-Scale changes in gene expression and in the chromatin landscape[ChIP-seq]

first: Gregory

last: Donahue

summary: Cellular senescence is a stable proliferation arrest in response to stress, associated with an altered secretory pathway (Senescence Associated Secretory Phenotype (SASP)). Senescence-associated proliferation arrest and the SASP are thought to act in concert to promote tumor suppression and tissue aging.  While chromatin regulation and down regulation of lamin B1 have been implicated as effectors of cell senescence, functional interactions between them are poorly understood.  We compared the genome-wide distributions of H3K4me3 and H3K27me3 between proliferating and senescent primary human cells and found dramatic differences, including large-scale domains of H3K4me3- and H3K27me3-enriched "mesas" and H3K27me3-depleted "canyons" in senescent cells.  Senescent mesas form at the sites of lamin B1-associated domains (LADs) in proliferating cells.  Mesas also overlap with regions that exhibit DNA hypomethylation in cancer, suggesting that chromatin changes in pre-malignant senescent cells foreshadow epigenetic changes in cancer.  Proliferating fibroblasts from Hutchinson-Gilford Progeria Syndrome patients expressing mutant lamin A (progerin) also show evidence of H3K4me3 mesas, suggesting a link between premature chromatin changes and accelerated cell senescence and tissue aging.  In contrast, canyons form mostly in between LADs and are enriched in genes, gene promoters and enhancers.  Strikingly, H3K27me3 loss in canyons is correlated with upregulation of key senescence genes, including genes comprising the SASP, indicating a link between global changes in chromatin structure and local regulation of gene expression.  Finally, premature reduction of lamin B1 in midlife proliferating cells triggers formation of senescence-associated mesas and canyons and accelerated senescence.  Together, our data illustrate a profound reorganization of chromatin during senescence, and suggest that down regulation of lamin B1 in senescence is a key trigger of global and local chromatin changes that impact gene expression, aging and cancer.

overall_design: One lane per sample, with replicates; four lanes H3 (one proliferating, one senescent, one control, one Ras-induced), four lanes H3K27me3 (three proliferating, one senescent), two lanes H3K4me3 (one control, one ras-induced)

last: Shah

first: Gabriel

last: Otte

first: Brian

last: Capell

last: Nelson

last: Cao

last: Aggarwala

first: Hazel

last: Cruickshanks

first: Taranjit

last: Rai

first: Tony

last: McBryan

last: Gregory

first: Davinder

last: Sandhu


title: Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros (RNA-seq: BCR-ABL transformed in vitro cell culture cells)

last: Schjerven

overall_design: RNA-Seq from BCR-ABL+ transformed cells at day 21 and day 28 of in vitro cell culture comparing wt, Ikaros-ZnF1-/- mutant and Ikaros-ZnF4-/- mutant.

last: Smale


title: Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros (ChIP-seq: Thymocytes)

overall_design: Ikaros ChIP-Seq from Whole Thymus comparing wt, Ikaros-ZnF1-/- mutant and Ikaros-ZnF4-/- mutant


title: Beta cell 5’-shifted isomiRs are candidate regulatory hubs in type 2 diabetes

city: Chapel Hill

first: Jeanette

last: Baran-Gale

state: NC

address: 120 Mason Farm Rd.

institute: University of North Carolina at Chapel Hill

summary: We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells cultured in 25mM glucose. We then developed and implemented an in-house short-read mapping strategy to analyze isomiR diversity.

overall_design: Profile of miRNA expression in MIN6 cells cultured in 25mM glucose.

first: Praveen

last: Sethupathy


title: Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros

overall_design: Refer to individual Series

first: Jami

last: McLaughlin

first: Seth

last: Frietze

first: Donghui

last: Cheng

first: Peggy

last: Farnham

first: Owen

last: Witte

first: Stephen


title: Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros (RNA-seq: Thymocytes)

first: Hilde

overall_design:
RNA-Seq from Whole Thymus comparing wt (3 replicates), Ikaros-ZnF1-/- mutant (2 replicates) and Ikaros-ZnF4-/- mutant (2 replicates)
RPKM_Thymocytes.txt (linked below as a supplementary file) reports the relative mRNA expression levels (RPKM)values for all annotated Refseq genes that had at least one read in at least one of the samples, with duplicates for the same gene (different transcripts for same gene) filtered out.  RPKM (Mortazavi et al., 2008) were calculated based on exonic reads obtained by using the software SeqMonk (Babraham Bioinformatics) and reference genome annotations from NCBI (mm9).


title: Supression of genetic recombination in the pseudoautosomal region and at subtelomeres in mice with a hypomorphic spo11 allele

city: Bethesda

first: Kevin

last: Brick

state: MD

address: 5/205 Memorial Drive

department: GBB

institute: NIDDK

type: Genome variation profiling by high throughput sequencing

summary: Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4.  We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes – normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.

overall_design: Detection of meiotic double strand breaks in mice with a hypomorphic Spo11 allele.

first: Fatima

last: Smagulova

first: Yongmei

last: Pu

first: Uttara

last: Sengupta

first: Rafael-Daniel

last: Camerini-Otero

first: Galina

last: Petukhova


title: mRNA-Seq of asi1-1, wild-type and MethlyC-Seq of asi1-1

status: Public on Sep 06 2013

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: 1, Using mRNA-Seq to get expression profiling of asi1-1 mutant and WT; 2, Using MethylC-Seq to provide single-base resolution of DNA methylation status in asi1-1 mutant

overall_design: mRNA-Seq: 2 samples examined, Col-0 wild-type with 35S-SUC2 transgene(termed as WT) and asi1-1 mutant (Col-0 background with 35S-SUC2 transgene); MethylC-Seq: 1 sample examined, asi1-1 mutant

first: Xingang

last: Wang

first: Cheng-Guo

last: Duan

first: Kai

last: Tang


title: MethlyC-Seq of ibm1-4

summary: Using MethylC-Seq to provide single-base resolution of DNA methylation status in ibm1-4 mutant

overall_design: 1 sample examined, ibm1-4 mutant


title: Involvement of microRNA-related regulatory pathways in glucose-mediated control of Arabidopsis early seedling development

city: Campinas

first: Renato

last: Vicentini

state: SP

address: Cidade Universitaria "Zeferino Vaz"

department: Centro de Biologia Molecular e Engenharia Genetica - CBMEG

country: Brazil

institute: Universidade Estadual de Campinas - UNICAMP

summary: In plants, sugars such as glucose act as signaling molecules that promote changes in gene expression programs impacting on growth and development.  Recent evidences have revealed the potential participation of mRNA decay control in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2) and miRNA-activity (ago1-25) were evaluated. Both mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways may be involved in the glucose-mediated delay of early seedling development. The expression profile of 200 primary miRNA transcripts (pri-miRs) were evaluated by large-scale quantitative real-time polymerase chain reaction (qRT-PCR) profiling revealing that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miRNA by glucose. These data indicate that deficiency in miRNA machinery leads to a deregulated expression of several miRNA target genes in response to glucose. Also, such deficiencies result in glucose-promoted misexpression of genes for the three Abscisic Acid signaling elements ABI3, ABI4 and ABI5. Thus, miRNA-regulatory pathways play a role in the adjustments of growth and development triggered by glucose signaling.

overall_design: Three samples, including untreated control, 2% glucose-treated and 2% mannitol-treated samples.

first: Gustavo

last: Duarte

first: Cleverson

last: Matiolli

first: Bikram

last: Pant

first: Armin

last: Schlereth

first: Wolf

middle: R

last: Scheible

first: Mark

last: Stitt

first: Michel

last: Vincentz


title: Genome-wide analysis of p53 binding by ChIP-seq

status: Public on Sep 05 2013

city: New Orleans

first: Yuwen

last: Li

address: 1430 Tulane Ave SL37 RM5534

department: Pediatrics

institute: Tulane University

summary: We used ChIP-seq to identify the genomic locations bound by p53 in mouse kidney

overall_design: Snap-frozen kidney was crosslinked with formaldehyde to generate a snap shot of all protein-DNA interactions occurring in the nucleus at that particular point in time, then antibodies were used to immunoprecipitate the p53 protein together with crosslinked DNA fragments. Following reversal of crosslinks and digestion of protein, adaptors were ligated to immunoprecipitated DNA and PCR was used to generate clonally amplified amplicons before sequencing by synthesis using the Illumina Solexa Genome Analyzer.

first: Jiao

last: Liu

first: Nathaniel

last: McLaughlin

first: Dimcho

last: Bachvarov

first: Zubaida

last: Saifudeen

first: Samir

last: El-Dahr


title: Architectural niche organization by LHX2 is linked to hair follicle stem cell function

city: New York

first: Alicia

last: Rodriguez-Folgueras

address: 1230 York Ave.

institute: Rockefeller University

summary: To identify direct LHX2 target genes in HFSCs, we performed chromatin immunoprecipitation and deep sequencing (ChIP-seq) analysis using FACS-isolated HFSCs.

overall_design: Three independent LHX2 ChIP-seq experiments were conducted.

last: Folgueras

first: Elaine

last: Fuchs


title: miR-146a-5p circuitry uncouples cell proliferation and migration, but not differentiation, in human mesenchymal stem cells

city: Taipei

first: Jui-Yu

last: Hsieh

address: No. 155, Sec 2, Li-Nong Street

country: Taiwan

institute: National Yang-Ming University

summary: Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton's jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared to BM-MSCs.

overall_design: One human BM-MSC (pooled sample of 4 independent donors), one human WJ-MSC (pooled sample of 3 independent donors), and differentiated osteocytes and adipocytes derived from BM-MSCs after 2 weeks induction.

first: Jui

middle: Y

first: Tse

last: Huang

first: Shu

last: Cheng

first: Wei

last: Lin

first: Tsung

middle: N

last: Tsai

first: Oscar

last: Lee

first: Hsei

middle: W

last: Wang


title: Integral nuclear pore proteins bind to Pol III genes and are required for Pol III transcript processing in C. elegans [seq]

city: Cincinnati

first: Kohta

last: Ikegami

state: OH

address: 240 Albert Sabin Way, Cincinnati, OH 45229

department: Division of Molecular Cardiovascular Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-ß IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans.

overall_design: Genome-wide ChIP-seq and ChIP-chip were performed in mixed-stage C. elegans embryos for nuclear pore proteins NPP-13, NPP-3, IMB-1 and chromatin proteins Pol III (RPC-1), TBP-1, TFC-1 (SFC-1), TFC-4 (TAG-315), and Pol II (AMA-1). For RPC-1 and TBP-1 ChIP-seq, embryos depleted for NPP-13 were also used. Total RNAs from wild-type, NPP-13 RNAi, and IMB-1 RNAi embryos were analyzed by RNA-seq.

first: Jason

last: Lieb


title: Genome-wide identification of molecular pathways and biomarkers in response to arsenic exposure in zebrafish liver

status: Public on Sep 04 2013

city: Singapore

first: Zhiyuan

last: Gong

address: Kent Ridge

country: Singapore

institute: National University of Singapore

summary: In the present study, we employed the RNA sequencing platform to examine the molecular response of zebrafish liver to arsenic exposure and carry out detailed transcriptomic analyses for further understanding of molecular toxicity. We found that several important biological processes were perturbed by arsenic exposure, including oxidation reduction, translation, iron ion transport, cell redox and homeostasis, as well as related pathways in metabolism and diseases. Furthermore, as there are currently no biomarker genes available for predicting arsenic exposure, we took the advantage of RNA sequencing platform to identify most suitable biomarker genes from top responsive genes to arsenic exposure. We first validated these top responsive genes by RT-qPCR in zebrafish and then in Japanese medaka (Oryzias latipes) at individual fish level for more robustly responsive genes across different fish species.

overall_design: Transcriptome profiling of arsenic-treated sample and control sample were generated by deep sequencing using 3' RNA-SAGE on the SOLiD system.

first: Hongyan

last: Xu

first: Siew

middle: H

last: Lam


title: De Novo Transcriptomic Analysis of Hydrogen Production in the Green Alga Chlamydomonas moewusii through RNA-Seq

city: Golden

first: Shihui

last: YANG

state: CO

address: 1617 Cole Blvd

department: National Bioenergy Center

institute: National Renewable Energy Lab

summary:
Background: Microalgae can make a significant contribution towards meeting global renewable energy needs in both lipid-based liquid biofuel and hydrogen biofuel. The development of energy-related products and chemicals from algae could be accelerated with improvements in systems biology tools, and recent advances in sequencing technology provide a platform for enhanced transcriptomic analyses. However, these techniques are still heavily reliant upon available genomic sequence data.  We have developed a de novo sequencing, annotation, and quantitation pipeline that can be applied to unsequenced organisms for effective quantitative gene expression profiling. Chlamydomonas moewusii is a unicellular green alga capable of evolving molecular hydrogen (H2) under both dark and light anaerobic conditions, and has high hydrogenase activity that can be rapidly induced. However, to date, there is no systematic investigation of transcriptomic profiling during induction of hydrogen photoproduction in this organism.
Results: In this work, we measured rates of hydrogen production and extracted RNA from samples of C. moewusii following various lengths of dark anaerobic incubation. RNA-Seq was applied to investigate transcriptomic profiles during the dark anaerobic induction of hydrogen photoproduction. One hundred fifty six million reads generated from seven samples were then used for de novo assembly after data trimming. The BlastX results against NCBI database and Blast2GO results were used to interpret the functions of the assembled 39,136 contigs, which were then used as the reference transcripts for RNA-Seq analysis. Nearly 98% transcripts had Blast hits, although more than one-third were annotated as hypothetical proteins. The expression value of RNA-Seq results was imported into statistical software for data quality control, normalization, and subsequent statistical analyses such as One-way ANOVA and K-means Clustering. Our results indicated that more transcripts were differentially expressed during the period of early and higher hydrogen photoproduction, and fewer transcripts were differentially expressed when rates of hydrogen photoproduction decreased.
Conclusions: Herein, we have described a workflow to analyze RNA-Seq data without reference genome sequence information, which can be applied to the rapid development of other unsequenced microorganisms (both prokaryotic and eukaryotic) with the potential for development as fuel production strains.  This study provided the first transcriptomic RNA-Seq dataset, as well as biological insights into the metabolic changes that occur concomitant with induction of hydrogen photoevolution in C. moewusii, which can help further development of this organism as a hydrogen photoproduction strain.

overall_design: Examine time course gene differential expression post anaerobic induction for hydrogen production

last: Yang

first: Michael

last: Guarnieri

first: Sharon

last: Smolinski

first: Maria

last: Ghirardi

first: Philip

last: Pienkos


title: Gene content or gene expression, which determines the difference in host specificity and virulence of strains of Xanthomonas citri subsp. citri?

city: Lake Alfred

first: Neha

last: Jalan

state: Florida

address: CREC, 700 Experiment station road

department: Deptarment of Microbiology and Cell Science

institute: University of Florida

summary: Xanthomonas is one important model microbe to study the molecular determinants of virulence and host range of pathogens since Xanthomonas is capable of infecting numerous monocotyledonous and dicotyledonous plants.  Among the plant diseases caused by Xanthomonas, X. citri subsp. citri (Xcc) causes citrus canker, which has significant impact on citrus production. Xcc is classified into different strains primarily by host range including A and Aw. The A (Asiatic) strain (XccA) has a wide host range and is most virulent, whereas Aw (Wellington) strain has restricted host range including Key or Mexican lime and alemow. We hypothesized that not only gene content but also gene expression contributes to the difference in virulence and host range of closely related strains. To test our hypothesis, comparative genomic and transcriptome analyses were conducted to study the two closely related Xcc A and Aw strains. The genome of X. citri subsp. citri strain Aw12879 (Xcaw) was completely sequenced using 454 Pyrosequencing, Illumina sequencing and Optical mapping. The finished genome (5.3 Mb chromosome and two plasmids pXcaw19 and pXcaw58) of Xcaw was annotated, curated and compared with XccA genome. Protein blast revealed multiple genes including type III secretion system (TIIISS) effectors xopAF and xopAG are present in Xcaw but absent in XccA. Comparative genomic analysis showed various changes in genes encoding LPS and type IV secretion system. Furthermore, RNA-Seq was used to compare expression profile of Xcaw and XccA in nutrient rich (NB) medium and XVM2 medium which is known to mimic the intercellular space of plant cells using Illumina sequencing.  Multiple avirulence/effector genes were over-expressed in Xcaw compared to XccA which might contribute to the limited host range of Xcaw compared to XccA.  The overexpression of genes involved in cell wall degradation, attachment, ROS (reactive oxygen species) scavenging, nutrient transportation in XccA might contribute to its expanding of host range. Our data suggest that both gene content and gene expression contribute to difference in virulence and host range of bacterial pathogens. This study lays the foundation to further characterize the mechanisms for virulence and host range of strains of X. citri subsp. citri and other bacterial pathogens.

overall_design: mRNA expression profiles of Xcc strain A and Aw were generated in 2 media: NB and XVM2 by deep sequencing, in triplicate, using Illumina GAII.

first: Nian


title: Transcriptome analysis of rice pathogen Acidovorax avenae subsp. avenae cultivated in vitro, in vivo and in co-culture with the rice rhizobacterium Burkholderia seminalis

status: Public on Sep 03 2013

city: HangZhou

first: Bin

last: Li

state: Zhejiang

address: 866-Yuhangtang Road

department: Biotechnology

institute: Zhejiang University

summary: Determining how a bacterial pathogen responds to its host and other bacterial species by altering gene expression is key to understand its pathogenesis and environmental adaption. Here, we used RNA-Seq to comprehensively and quantitatively assess the transcriptional response of the rice bacterial pathogen Acidovorax avenae subsp. avenae strain RS-1 cultivated in vitro, in vivo and in co-culture with rice rhizobacterium Burkholderia seminalis R456. Results revealed a surprisingly large number of regulatory differences between these conditions indicating adaptation of A. avenae subsp. avenae to specific ecological conditions. In particular, a number of potential virulence factors such as type 3 secretion system proteins were specifically expressed under in vivo conditions, whereas genes whose protein products are involved in inter-bacterial interaction such as auxin efflux carrier, small mechanosensitive ion channel protein, and ureidoglycolate hydrolase were among those specifically up-regulated under co-culture conditions. In addition, global genomic analysis of strain RS-1 identified 406 putative non-coding (nc) RNA genes. Interestingly, 8 ncRNA genes that were uniquely expressed under in vivo may be linked to pathogenicity while 4 ncRNA genes that were uniquely expressed under coculture conditions may be involved in adaption to co-cultivation with B. seminalis. Expression data obtained by RNA-Seq were also confirmed for selected genes by quantitative real-time PCR and two-dimensional gel electrophoresis as well as knockout analysis.

overall_design: Aaa strain RS-1 and B. seminalis strain R456 was isolated from diseased rice plants (Li et al., 2011; Xie et al., 2011) and rice rhizosphere (Zhang et al., 2007; Li et al., 2011), respectively, in our previous studies, and were stored in 20-30% sterile glycerol at -80°C. The samples of Aaa strain RS-1 for in vitro and in vivo analysis were prepared as described before (Ibrahim et al., 2012). The co-culture analysis of Aaa strain RS-1 with B. seminalis strain R456 were conducted according to Ruiz et al. (2009) and Di Cagno et al. (2009). Briefly, Aaa strain RS-1 and B. seminalis strain R456 was inoculated and incubated in chambers of a double culture vessel apparatus separated by a 0.4-μm membrane filter (Millipore Isopore™). In order to avoid the possible contamination during in vivo and co-culture operation, all bacterial samples were further confirmed based on the sequence analysis of 16S-rRNA (Li et al., 2011). Then samples were processed for RNA harvesting, mRNA purification and cDNA synthesis.

first: Muhammad

last: Ibrahim

first: Mengyu

last: Ge

first: Zhouqi

last: Cui

last: Kube

first: Yanli

first: Fei

first: Gongyou

last: Chen

first: Guanlin

last: Xie

first: Guochang

last: Sun

first: Zhonghua

last: Ma


title: Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone 4 (H4) Epigenome

city: Rochester

first: Jennifer

last: Westendorf

address: 200 1st St SW

institute: Mayo Clinic

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


title: Chromatin Immunoprecipitation of Nuclear Pore Proteins in C. elegans

first: Kohta

last: Ikegami

type: Genome binding/occupancy profiling by genome tiling array


title: The developmental patterning of 5-hydroxymethylcytosine during neuronal differentiation

first: Michael

last: Brainard

address: 675 Nelson Rising Ln

institute: University of California, San Francisco

summary: Recent analyses of 5-hydroxymethylcytosine (5hmC) suggest that the modified base influences transcription by acting as an intermediate to demethylation. However, elevated levels of 5hmC within neural tissues suggest that the mark is stable and may directly contribute to gene regulation. Here, we characterize the in vivo genomic patterning and stability of 5hmC in three defined developmental states of the mouse main olfactory epithelium – multipotent stem cells, neuronal progenitors, and mature olfactory sensory neurons. 5hmC is globally enriched in neuronal progenitors and neurons relative to the stem population with significant enrichment on the bodies of transcriptionally active genes and intergenic enhancers. Although gene body 5hmC levels are positively correlated with cell type-specific gene expression in all developmental stages, genes that are commonly expressed within both neuronal progenitors and mature neurons experience the greatest increases in 5hmC levels. Moreover, enriched levels of the modified base on intergenic conserved regions correlate with cell type-specific expression of nearby genes and coincide with developmentally regulated enhancer-promoter interaction at one locus examined. Finally, an analysis of two-month old neurons indicates that elevated 5hmC levels persist in fully differentiated neurons. Together, these data suggest that 5hmC stabilizes the expression of developmentally active genes through effects at both gene bodies and intergenic regulatory elements.

overall_design: 9 Samples

first: Bradley

middle: M

last: Colquitt

first: Stavros

last: Lomvardas


title: Gene body 5-hydroxymethylcytosine facilitates transcription in olfactory neurons

summary: The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene function and cellular identity. To address this issue in an in vivo neuronal population, we assessed the patterning, stability, and function of the base within gene bodies in olfactory sensory neurons. We find that gene body 5hmC linearly correlates with transcriptional output and is stable in fully mature neurons and those lacking de novo methyltransferase activity. Overexpression of Tet3, which oxidizes methylated cytosines (5mC) to 5hmC, markedly alters gene body 5hmC levels and provides evidence that 5hmC facilitates transcription. This manipulation disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system that are necessary for proper physiology. Our results support a direct, positive and physiologically significant role for gene body 5hmC in transcriptional elongation and the maintenance of cellular identity independent of its function as an intermediate to demethylation.

overall_design: We assessed the role of 5hmC in mature olfactory sensory neurons by assessing 5hmC levels in 2 month old neurons, olfactory epithelia lacking Dnmt3a, and mOSNs overexpressing Tet3. To determine genome-wide levels of 5hmC, we performed DNA immunoprecipitation coupled to Illumina sequencing. To determine transcript levels, we prepared and sequenced rRNA-depleted cDNA libraries.


title: Deep sequencing of exosomal and cellular miRNAs in MDA-MB-231 and MCF-10A cells

status: Public on Sep 01 2013

city: Duarte

first: Xiwei

last: Wu

address: 1500 E Duarte Rd

department: Molecular and Cellular Biology

institute: Beckman Research Institute

summary: To identify the miRNAs that are differentially expressed and secreted between the MDA-MB-231 metastatic breast cancer cells and the MCF-10A non-cancerous human mammary epithelial cells, we profiled the cellular and exosomal small RNAs (between 17 and 52 nt) isolated from these two cell lines by Solexa deep sequencing.  MiRNAs that are significantly different between the two cell lines are identified.

overall_design: RNA was extracted from cultured MDA-MB-231 and MCF-10A cells or purified exosomes secreted by these cells, and subjected to library construction and Solexa deep sequencing.

first: Shizhen

last: Wang


title: Inducible knockdown of Plasmodium gene expression using the glmS ribozyme

city: Pathumthani

first: Philip

middle: James

last: Shaw

state: Pathumthani

address: 113 Phahonyothin Road

department: National Center for Genetic Engineering and Biotechnology

country: Thailand

institute: National Science and Technology Development Agency

summary: Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region (3′-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery.

overall_design: mRNA profiles were generated from 3D7 wild-type and DHFR-TS-GFP_glmS integrant parasites in untreated and  treated with 10 mM Glucosamine conditions in duplicate.


title: The effects of dietary selenium on selenocysteine incorporation and selenoprotein expression

status: Public on Sep 17 2013

city: Salt Lake City

last: Howard

state: UT

department: Human Genetics

institute: University of Utah

summary: The goal of this study was to determine the effects of dietary selenium levels on translational control of selenoprotein synthesis in mouse liver.

overall_design: Wild type mice and mice expressing a mutant Sec-tRNA gene (TrspA37G) were fed diets supplemented with 0, 0.1, or 2 ppm selenium for 6 weeks. Livers were harvested and ribosome and mRNA profiles were generated by deep-sequencing using the Illumina HiSeq 2000.


title: Drosophila Gtsf1 is an essential component of the Piwi mediated transcriptional silencing complex

city: Vienna

first: Grzegorz

last: Sienski

address: Dr. Bohr-Gasse 3

country: Austria

institute: IMBA-Institute of Molecular Biotechnology of Austrian Academy of Sciences

summary: The PIWI interacting RNA pathway is a small RNA silencing system that keeps selfish genetic elements such as transposons under control in animal gonads. Several lines of evidence indicate that nuclear PIWI family proteins guide transcriptional silencing of their targets, yet the composition of the underlying silencing complex is unknown. Here we demonstrate that the double CHHC zinc finger protein Gtsf1 is an essential factor for Piwi mediated transcriptional repression in Drosophila. Cells lacking Gtsf1 contain nuclear Piwi loaded with piRNAs, yet Piwi's silencing capacity is ablated. Gtsf1 interacts stably with a sub-population of nuclear Piwi and loss of Gtsf1 phenocopies loss of Piwi in terms of deregulation of transposons, loss of H3K9me3 marks at euchromatic transposon insertions and deregulation of genes in proximity to repressed transposons. We propose that only a small fraction of nuclear Piwi interacts productively with a target RNA, resulting in assembly of a silencing complex with Gtsf1 as one core component.

overall_design: impact of loss of DmGtsf1 on transcription and H3K9m3 in ovarian somatic cells (OSC)

first: Julius

last: Brennecke


title: Genome-wide Analysis of Transcriptional Regulators in Human Blood Stem/Progenitor Cells reveals a densely interconnected network of coding and non-coding genes.

city: Sydney

first: Jason

last: Wong

state: NSW

address: Level 2, Lowy Cancer Research Centre, University of New South Wales

institute: UNIVERSITY OF NEW SOUTH WALES

summary: Combinatorial transcription factor (TF) interactions regulate hematopoietic stem cell formation, maintenance and differentiation, and are increasingly recognised as drivers of stem cell signatures in cancer.  However, genome-wide combinatorial binding patterns for key regulators do not exist in primary human hematopoietic stem/progenitor cells (HSPCs) and have constrained analysis of the global architecture of the molecular circuits controlling these cells.  Here we provide new high-resolution genome-wide binding maps of seven key TFs (FLI1, ERG, GATA2, RUNX1, SCL, LYL1 and LMO2) in human CD34+ HSPCs together with quantitative RNA and microRNA expression profiles.  We catalogue binding of TFs at coding genes and microRNA promoters and report that combinatorial binding of all seven TFs is favoured and is associated with differential expression of genes and microRNA in HSPCs.   We also uncover a hitherto unrecognized association between FLI1 and RUNX1 pairing in HSPCs, establish a correlation between the density of histone modifications, which mark active enhancers and the number of overlapping TFs at a peak and identify complex relationships between specific miRNAs and coding genes regulated by the heptad.  Taken together, this study demonstrates that a heptad of TFs forms a dense auto-regulatory core in human HSPCs with binding of all seven TFs at tissue specific regulatory elements of heptad genes and collectively regulates miRNAs that in turn target components of the heptad and genes regulated by the heptad.

overall_design: Examination of cominatorial binding by 7 transcription factors, 1 IgG control along with mRNA and small RNA sequencing in human CD34+ cells

first: John

last: Pimanda

first: Dominik

last: Beck


title: Distribution of insulator proteins CP190, dCTCF, Su(Hw) and Mod(mdg4)2.2 in Drosophila S2 cells in the presence or absence of dPARP inhibitor 3AB

status: Public on Sep 15 2013

first: Chin-Tong

last: Ong

address: 1 Research Link

department: Developmental Biology

institute: Temasek Life Sciences Lab

summary: We mapped the localization of insulator proteins in Drosophila S2 cells in the presence or absence of 12mM of 3AB. We found that inhibition of PARylation affects DNA binding of insulator proteins only at a small subsets of genomic sites.

overall_design: Examination of genomic occupancy for insulator proteins in S2 cells with or without 3AB inhibition.

first: Victor

last: Corces

first: Chintong

first: Kevin

last: Van Bortle


title: Genome-wide approaches reveal functional VEGF-inducible NFATc1 binding to the angiogenesis-related genes in endothelium

city: Mitaka

first: Jun-ichi

last: Suehiro

state: Tokyo

address: 6-20-2, Shinkawa

department: Department of Pharmacology and Toxicology

country: Japan

institute: Kyorin University School of Medicine

overall_design: Refer to individual Series


title: Global analysis of alternative splicing regulated by RBM10

city: Gießen

first: Andreas

last: Gogol-Döring

address: Wiesenstraße 14

department: MNI

country: Germany

institute: Technische Hochschule MIttelhessen

summary: RBM10 is an RNA binding protein that was identified as a component of spliceosome complex, suggesting its potential role in splicing regulation. However, the direct experimental evidence for this function has been lacking. Here we characterized in vivo RBM10-RNA interactions and investigated the role of RBM10 in splicing regulation at the global level. We observed significant RBM10-RNA interactions in the vicinity of splice sites and identified hundreds of splicing changes following perturbation of cellular RBM10 abundance. A RNA splicing map integrating the binding pattern and splicing profiles revealed a significant correlation between RBM10-enhanced exon skipping events and its binding close to the splicing sites of both upstream and downstream introns. Furthermore, we demonstrated the splicing defects in a patient carrying a RBM10 mutation. Overall, our data provided insights into the mechanistic model of RBM10-mediated splicing regulation and established genomic resources for future studies on its function  in different pathophysiological contexts.

overall_design: We sequenced the mRNA of HEK293 cells and LCL cells, and we determined the RBM10 binding sites using PARCLIP in HEK293 cells. In total we sequenced four mRNA-Seq libraries for KD and two for OE in HEK293 cells; for each of these libraries, we also sequenced one control library. We also sequenced the mRNA of one patient LCL and two normal LCL libraries. Two replicates of PARCLIP sequencing were perfomed.

first: Yongbo

first: Hao

last: Hu

first: Sebastian

last: Fröhler

first: Marvin

last: Jens

first: Jonas

last: Maaskola

first: Yunxia

last: Ma

first: Yasuhiro

last: Murakawav

first: Claudia

last: Quedenau

first: Markus

last: Landthaler

first: Vera

last: Kalscheuer

first: Yuhui

first: Wei

last: Chen


city: Stockholm

first: Rickard

last: Sandberg

address: Berzelius vag 35

department: Department of Cell and Molecular Biology

country: Sweden

institute: Karolinska Institutet

summary: Improved Smart-Seq for sensitive full-length transcriptome profiling in single cells.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Cells of four different origins were profiled using commercial SMARTer and compared to five variants of an improved protocol (Smart-Seq2).

first: Simone

last: Picelli

first: Åsa

last: Björklund


title: Analysis of banana transcriptome and global gene expression profiles in banana roots in response to infection by Race 1 and Race 4 of Fusarium oxysporum f. sp. Cubense

city: Houston

first: Jiaofang

last: Shao

state: Texas

address: 6431 Fannin St

department: Department of Biochemistry and Molecular Biology

institute: McGovern Medical School, University of Texas Health Science Center

summary:
Background: Banana (Musa) is one of the most important crops grown in tropical and sub-tropical areas. Cavendish, the most widely grown banana cultivar, is a triploid derived from an intra-species cross. Cavendish is relatively resistant to Race 1 of Fusarium oxysporum f. sp. Cubense (Foc1) which caused wide spread Panama disease during 1960s but is susceptible to Race 4 of Foc (Foc4) which has been causing epidemics in large areas of banana fields in Asia and Australia in the last decade and is threatening world banana production. The genome of the diploid species Musa acuminata (AA) which is the ancestor of a majority of cultivated banana has recently been sequenced. Availability of banana transcriptomes will be highly useful for improving banana genome annotation and assembly and for banana biological research. The knowledge of global gene expression patterns influenced by infection by different Foc races will help to understand the pathogenesis processes and the host responses to the infection.
Results: RNA samples extracted from different organs of the Cavendish cultivar were pooled for deep sequencing using the Illumina sequencing technology. The assembled reads were aligned with the genome of M. accuminata and with sequences in the Genbank databases. The analysis led to identification of 842 genes that were not annotated by the Musa genome project. A large number of simple nucleotide polymorphisms (SNPs) and short insertions and deletion (indels) were identified from the transcriptome data. GFP-expressing Foc1 and Foc4 was generated and used to monitor the infection process. Digital gene expression (DGE) profiling analysis was carried out to obtain transcriptome profiles influenced by infection with Foc1 and Foc4 in banana roots at 3, 27, and 51 hours post-inoculation. Both Foc1 and Foc4 were found to be able to invade banana roots and spread to root vascular tissues in the first two days following inoculation. The profiling analysis revealed that inoculation with Foc1 and Foc4 caused similar changes in the gene expression profiles in the infected banana roots. The Foc infection led to induction of many well-known defense-related genes including PATHOGENESIS-RELATED 5 (PR5), PAL, and a lignin-forming peroxidase. The WRKY40 gene, which is a negative regulator of the defense pathway in Arabidopsis, was quickly and strongly suppressed by the infection. Two genes encoding the ethylene biosynthetic enzyme ACC oxidase and several ethylene-responsive transcription factors were among strongly induced genes by both Foc1 and Foc4
Conclusions: Both Foc1 and Foc4 are able to spread into the vascular system of banana roots during the first two days of the infection process and their infection led to similar gene expression profiles in banana roots. The transcriptome profiling analysis indicates that the ethylene synthetic and signalling pathways were activated in response to the Foc infection.

overall_design: Digital gene expression (DGE) profiling analysis was carried out to obtain transcriptome profiles influenced by infection with Foc1 and Foc4 in banana roots at 3, 27, and 51 hours post-inoculation. The plants whose roots were immersed in the culture medium without the pathogen (mock inoculation) were used as a control.

first: Chunqiang

last: Li

first: Yejun

last: Wang

first: Wenbin

first: Dianjing

last: Guo

first: Bin

last: Yan

first: Yiji

last: Xia

first: Ming

last: Peng


title: Global analysis of NFATc1 binding and histone marks in VEGF-activated endothelial cells

first: Jun-ichi

last: Suehiro

summary: We performed the newly mapping of genome-wide NFATc1 binding events in VEGF-stimulated primary cultured endothelial cells, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Combined NFATc1 ChIP-seq profile and the epigenetic histone marks revealed that predominant NFATc1-occupied peaks were overlapped with promoter marking but not silencer marking. DNA microarrays with NFATc1 expression or knockdown indicated the predominant NFATc1 binding targets were correlated with induced patterns.

overall_design: Examination of NFATc1 and two different histone marks in HUVEC in the presence/absense of VEGF.

first: Yasuharu

last: Kanki

first: Mai

last: Miura

first: Yuuka

last: Manabe

first: Hiroyuki

last: Aburatani

first: Tatsuhiko

last: Kodama

first: Takashi

last: Minami


title: ChIP-seq in oat-maize additional lines using CenH3 antibody

city: Madison

first: Yufeng

last: Wu

state: WI

address: 1575 Linden Drive

institute: UW-Madison

summary: Many existing centromeres may have originated as neocentromeres that activated de novo from non-centromeric regions. However, the evolutionary path from a neocentromere to a mature centromere has been elusive. Here we analyzed the centromeres of six chromosomes that were transferred from maize into oat as the result of an inter-species cross. Centromere size and location were assayed by chromatin immunoprecipitation for the histone variant CENH3, which is a defining feature of functional centromeres. Maize and oat are highly divergent and differ in genome size by four fold. Two isolates of maize chromosome proved to contain neocentromeres in the sense that they had moved from the original site, whereas the remaining seven centromeres (1, 2, 5, 6, 8, 9 and 10) were retained in the same area in both species. In all cases the CENH3-binding domains were dramatically expanded to encompass a larger area in the oat background (~4 Mb) than the average centromere size in maize (~2 Mb). The expansion of maize centromeres appeared to be restricted by the transcription of genes located in regions flanking the original centromeres. The results from the current study provide evidence that (1) centromere size is regulated; (2) centromere sizes tend to be uniform within a species regardless of chromosome size or origin of the centromere; and (3) neocentromeres emerge and expand preferentially in gene poor regions. Our results, together with data from several animal species, suggest that centromere size expansion may be a key factor in the survival of neocentric chromosomes in natural populations.

overall_design: Using ChIP-seq with CenH3 antibody, we defined the functional centromere regions in  9 oat-maize additional lines. Two oat maize additional lines include maize chromosome 3 in oats background (OMA3.01 and neoM3). The other lines include maize chromosome 1,2,5,6,8,9, and 10, respectively.

first: Kai

first: Wenli

last: Zhang

first: R

last: Dawe

first: Jiming

last: Jiang


title: The tetraspanin CD9 affords high purity capture of all murine hematopoietic stem cells.

city: Lund

first: Shamit

last: Soneji

address: Sölvegatan 19

department: Biomedicinskt Centrum

institute: Lund University

summary: Prospective isolation is critical to understand the cellular and molecular aspects of stem cell heterogeneity. Here we identify the cell surface antigen CD9 as a novel positive marker that provides a simple alternative for hematopoietic stem cell-isolation at high purity

overall_design: mRNA profiles of LT and ST HSCs

first: Goran

last: Karlsson


title: Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone 4 (H4) Epigenome (ChIP-Seq)

first: Jennifer

last: Westendorf

summary: ChIP-Seq analysis revealed that suberoylanilidehydroxamic acid (SAHA) increases genome-wide H4 acetylation in differentially regulated genes, except for the 500 bp upstream of transcription start sites (TSS).

overall_design: Chromatin immunoprecipitation (ChIP) with massively parallel high throughput sequencing (Seq) was used to map genome-wide histone H4 acetylation (K4/7/11/15) in the presence or absence of SAHA in differentiating  MC3T3 sc4 osteoblasts.

first: Amel

last: Dudakovic

first: Jared

last: Evans

first: Ying

last: Li

first: Sumit

last: Middha

first: Meghan

last: McGee-Lawrence

first: Andre

last: van Wijnen


title: Genome-wide analysis of miRNA-mRNA interactions in the marrow microenvironment

city: Aurora

first: Manoj

last: Pillai

address: Building 500 - 13001 E. 17th Place, Campus Box C290, Room E1354

institute: University of Colorado

type:
Other
Non-coding RNA profiling by array


title: Identification of expressed and conserved human non-coding RNAs

city: Aarhus

first: Morten

middle: Muhlig

last: Nielsen

address: Palle Juul-Jensens Bouleward 99

department: Department of Molecular Medicine

country: Denmark

institute: Aarhus University

summary:
Methods: RNA profiles of 12 tissues were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks.
Results: This work has focused on identification of non-coding RNAs expressed in human tissues. We have identified a set of non-coding RNAs that are both expressed and conserved, as described in the manuscript accompagnying this work.

overall_design: total RNA profiles of 12 normal human tissues, no replicates.

first: Jakob

last: Pedersen


title: Non-IG Aberrations of FOXP1 in B-Cell Malignancies Lead to an Aberrant Expression of N-Truncated Isoforms of FOXP1

city: Leuven

first: Julio

last: Finalet

address: Herestraat 49

country: Belgium

institute: KULeuven

summary: The transcription factor FOXP1 is implicated in the pathogenesis of B-cell lymphomas through immunoglobulin heavy chain (IGH) locus-related chromosomal translocations leading to dysregulated expression of FOXP1. Translocations of FOXP1 with non-IG gene sequences have been also reported, but the molecular consequences of such aberrations remain undetermined. Here, using molecular cytogenetics and molecular biology studies, we comprehensively analyzed four lymphoma cases with non-IG rearrangements of FOXP1 and compared these with cases harboring t(3;14)(p13;q32)/IGH-FOXP1 and FOXP1-expressing lymphomas without underlying t(3p13/FOXP1). We found that non-IG rearrangements are usually acquired during evolution of lymphoma and constantly target the coding region of FOXP1, promiscuously fusing with coding and non-coding gene sequences at various reciprocal breakpoints (2q36, 10q24 and 3q11). Intriguingly, these rearrangements do not generate functional chimeric genes but commonly disrupt the full-length FOXP1 transcript leading to an aberrant expression of N-truncated FOXP1 isoforms, as shown by QRT-PCR and Western blot analysis. In contrast, cases with t(3;14)(p13;q32)/IGH-FOXP1 overexpress the full-length FOXP1. Collectively, our findings point to a dual mechanism through which FOXP1 is implicated in B-cell lymphomagenesis. The primary t(3;14)(p13;q32)/IGH-FOXP1 produces the full-length protein with potent oncogenic activity, whereas the secondary non-IG 17 rearrangements of FOXP1 generate N-truncated FOXP1 isoforms, likely driving progression of disease.

overall_design: Using molecular cytogenetics and molecular biology studies (including RNA-seq), we comprehensively analyzed four lymphoma cases with non-IG rearrangements of FOXP1 and compared these with cases harboring t(3;14)(p13;q32)/IGH-FOXP1 and FOXP1-expressing lymphomas without underlying t(3p13/FOXP1).

first: Leila

last: Rouhigharabaei

middle: A

last: Finalet Ferreiro

first: Thomas

last: Tousseyn

first: Jo

last: van der Krogt

first: Natalie

last: Put

first: Eugenia

last: Haralambieva

first: Carlos

last: Graux

first: Brigitte

last: Maes

first: Carmen

last: Vicente

first: Peter

last: Vandenberghe

first: Jan

last: Cools

first: Iwona

last: Wlodarska


title: Integration of epigenetic and transcriptional mechanisms governs functional commitment versus plasticity of gd T cell subsets

city: Almada

first: Ana

middle: Rita

last: Grosso

address: Campus de Caparica

department: Departamento Ciências da Vida

country: Portugal

institute: Unidade de Ciências Biomoleculares Aplicadas (UCIBIO)/Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa

summary: gd T cells are major innate sources of interleukin-17 (IL-17) and interferon-g (IFN-g), which are differentially produced by two thymically-derived subsets segregated on CD27 expression. However, the molecular mechanisms that program the functional differentiation of gd cells remain incompletely understood. Here we show that CD27+ gd cells are epigenetically committed to express Ifng but not Il17, whereas CD27- gd cells spontaneously make IL-17 but can be induced to produce IFN-g under specific inflammatory conditions. This “plastic” behavior of CD27- gd cells associates with permissive histone H3 marks at loci encoding Ifng and upstream “type 1” transcription factors. By contrast, Il17 and related “type 17” factors are epigenetically and transcriptionally active in CD27- but silenced in CD27+ gd cells. Hence, stable versus plastic behaviors of gd cell subsets are controlled by integrated epigenetic and transcriptional mechanisms that regulate the expression of “master” transcription factors and effector cytokine genes.

overall_design: ChIP was carried out on FACS-sorted cells from pooled spleen/ lymph nodes. The following antibodies were used: anti-histone H3K4me2 (07-030, Millipore) and anti-histone H3k27me3 (07-449, Millipore). Between 105 - 106 cells were crosslinked with formaldehyde and nuclei were isolated and sonicated with a Sanyo Soniprep 150 at an amplitude of 10 microns with 17 times 10s bursts, resulting in 200–400bp chromatin fragments. IP was carried out as previously described 49. The Immunoprecipitated DNA released from crosslinked proteins was extracted with the QiaQuick kit (Qiagen) in accordance with the manufacturer´s instructions. Deep sequencing was performed at the GeneCore facility of EMBL (http://www.genecore.embl.de/). At least 1 ng of immunoprecipitated DNA was used for library preparation according to the Illumina protocol.

first: Nina

last: Schmolka

first: Karine

last: Serre

first: Margarida

last: Rei

first: Daniel

last: Pennington

first: Anita

middle: Q

last: Gomes

first: Bruno

last: Silva-Santos


title: Tissue-specific methylomes reveal epigenetic memory in adult mouse tissue

city: Dallas

first: Gary

middle: Chung

last: Hon

state: TX

address: 5323 Harry Hines Blvd.

department: OB/GYN

country: USA

institute: UT Southwestern

summary: Cytosine methylation of DNA is an evolutionarily conserved mechanism from plants to animals with crucial roles in gene regulation. However, the variation between methylomes of normal tissues is largely unexplored. To better understand the epigenetic variation of a normal individual, we profiled DNA methylation using whole genome bisulfite sequencing in 17 tissues isolated from an individual mouse. We observed a unique distribution of CpG methylation for each tissue, which cluster based on cell lineage. Global analysis identified only one-eighth of the genome as tissue-specifically methylated. Remarkably, the vast majority of these regions exhibit hallmarks of cis-regulatory activity. Our results also reveal a novel class of dormant enhancers in adult tissues which retain an epigenetic memory of regulatory elements active during development. Together, these results expand the repertoire of regulatory information encoded within the methylome, and suggest mapping it as an alternative method to identify cell-type specific regulatory elements.

overall_design: whole genome bisulfite sequencing of mouse adult tissue

first: Bing

last: Ren


title: Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline

city: New Haven

first: Mengjie

last: Chen

address: 13 Pleasant St

institute: Yale University

summary: Piwi proteins and their associated piRNAs are essential in the germline where they repress transposition, regulate translation, and guide epigenetic programming.  Little is known, however, about the molecular mechanisms through which Piwi proteins and piRNAs mediate these processes.  Here, we show that an evolutionarily conserved Tudor and KH-domain containing protein, Tdrkh (a.k.a. Tdrd2), partners with Miwi and Miwi2 in mice via symmetrically dimethylated arginine residues in Miwi and Miwi2.  Tdrkh is localized to pi-bodies and piP-bodies and is required for nuclear localization of Miwi2.  Genetic deletion of Tdrkh arrests meiosis at the zygotene stage, demethylates Line1 DNA, and up-regulates Line1 transposition, but does not promote apoptosis.  Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs.  Specifically, in Tdrkh mutants, piRNAs accumulate as a distinct population of 5’U-containing 31-37nt RNA that largely complements the missing mature piRNAs.  These results demonstrate that the primary piRNA biogenesis pathway involves 3à5’ processing of the 31-37nt intermediates and that Tdrkh is required for this final step of piRNA biogenesis.   However, Tdrkh is not required for the secondary piRNA biogenesis pathway (i.e., the ping pong cycle).  These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.

overall_design: Tdrkh-floxed mice were generated by the University of Connecticut Gene Targeting and Transgenic Facility.  The targeting vector utilized C57Bl6 Tdrkh genome sequences from BAC clone RP23-263K17 (Chori BACPAC) and floxed exons 2-4 (the start codon is in exon 2) and was electroporated into D2 ES cells, a male hybrid C57Bl6/129SEV line.  Clones that survived positive selection with G418 and negative selection with gancyclovir were expanded and screened by PCR with primers recognizing endogenous and exogenous (vector-derived) sequences.  Positive clones were fused to CD-1 embryos, and germline transmission from resulting pups was confirmed by test crosses to CD-1 mice (which also confirmed that all gametes were derived from the targeted clones).  Positive animals were bred to FLP mice to delete the neomycin-targeting cassette, resulting in Tdrkh cKO mice on a C57Bl6/129 background.  cKO mice were bred with EIIa-Cre transgenic mice to excise the floxed exons 2-4 and generate Tdrkh +/- animals.  Heterozygous animas were intercrossed to remove the EIIA transgene. Two independent knockout lines were generated from independent cKO founder lines and showed identical phenotypes in all experiments performed.

first: Haifan

last: Lin

first: Jonathan

last: Saxe


title: Transcription profiling of Arabidopsis plants overexpressing SsCBF1

city: Harbin

first: Lili

last: Zhang

state: Hei Longjiang

address: Mucai street

country: China

institute: Northeast Agricultural University

summary: Solanum lycopersicoides (LA2408), collected at higher altitudes (up to 3600 meters) than any of other solanum species, is a wild nightshade distant-allied to cultivated tomato. Many traits of Solanum lycopersicoides including cold tolerance, resistance to virus diseases and insect pests were previously confirmed. Thus, it is an ideal candidate plant for isolating cold-tolerance-related genes. In this study, we successfully cloned the full-length cDNA of the CBF1 gene from Solanum lycopersicoides which was designated as SsCBF1. In order to investigate the possible functions of SsCBF1 in plant growth and stress responses, we generated transgenic Arabidopsis overexpressing SsCBF1. We employed the RNA-seq approach to identify the differentially expressed genes between the two genotypes. Processing of RNA samples on the Illumina HiSeq 2000 system produced more than 20 million reads, each 100bp in length, encompassing 2.0 Gb of sequence data for each sample which was then mapped to the reference genome. The statistical analysis identified a total of 338 differentially expressed genes between Col-0 (WT) and transgenic Arabidopsis overexpressing SsCBF1 with the criteria of Q-value < 0.001 and fold change >2, among which 120 (35.5%) were up-regulated and 218 (64.5%) were down-regulated.

overall_design: RNA-sequencing was carried out using one transgenic line (35S:SsCBF1-11) and the Col-0 (WT) plants. Total RNA was isolated with Trizol reagent (Invitrogen, USA) from the aerial parts of 4-week-old seedlings grown in parallel under unstressed conditions. Materials from 20 plants of each genotype were pooled for RNA isolation.


title: RNA sequencing in fly heads to examine the effect of spermidine feeding on transcription in the ageing fly brain.

city: Nijmegen

first: Jamie

last: Kramer

address: Geert Grootplein 10

country: Netherlands

institute: Radboud University Nijmegen Medical Centre

summary: mRNA sequencing was used to identify genome wide transcriptional changes occuring in fly heads in response to spermidine feeding. This study shed light on the molecular mechanisms through wich spermidine can protect against age-dependent memory impairment.

overall_design: mRNA profiles from 3 and 10 day old Drosophila melanogaster heads were generated in duplicate by deep sequencing using Illumina GAIIx. mRNA profiles from flies that were fed food with 5mM spermidine were compared to profiles from flies that had no spermidine in thier food.

first: Tom

middle: S

last: Koemans

first: Annette

last: Schenck

first: Varun

middle: K

last: Gupta

first: Lisa

last: Scheunemann

first: Tobias

last: Eisenberg

first: Christoph

last: Mettke

first: Sara

last: Mertel

first: Anuradha

last: Bhuke

first: Hendrik

last: Stunnenberg

first: Frank

last: Sinner

last: Magnes

first: Shubham

last: Dipt

first: Andre

last: Fiala

first: Martin

last: Schwaerzel

last: Madeo

first: Stephan

last: Sigris


title: Tnni2del175k mutant mice and their wild-type littermates using mRNA array and ChIP-seq

city: beijing

first: xiaoquan

last: zhu

address: N01 dahua road dongdan beijing

institute: beijing hospital

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


title: tnni2 Binding Genome Seqences

summary: ChIP-Seq analysis of the potential accumulated binding sites on whole genome by wild-type tnni2 and tnni2 del175k mutant

overall_design: The primary osteoblasts from Tnni2del175k/175k mice and wild-type littermates (n=3) were cultured for ChIP-Seq. ChIP and construct libraries were performed using the antibody against tnni2 (Santa Cluz Biotechnology) and conducted as previously described above. Illumina HiSeq 2000 is used for sequencing. Raw reads were aligned with the mouse reference 8 using Bowtie software and viewed with the IGV browser.

first: shaorong

last: gao

first: Ze

last: yang


title: Effect of L-Proline on mouse embryonic stem cells (ESCs)

status: Public on Aug 31 2013

city: Munich

first: Miriam

last: Gagliardi

state: Bayern

address: Kraepelinstrasse 2-10

institute: Max Planck Institute of Psychiatry

summary: We found that the non-essential amino acid L-proline (L-Pro) acts as a signaling molecule that promotes the conversion of embryonic stem cells (ESCs) into mesenchymal-like, spindle-shaped, highly motile, invasive pluripotent stem cells.This embryonic stem cell-to-mesenchymal-like transition (esMT) is accompanied by a genome-wide remodeling of the H3K9me3 and H3K36me3 histone marks.

overall_design: Examination of 2 different histone modifications in untreated ESCs and L-Pro treated ESCs

first: Claudia

last: Angelini

first: Maria

last: Matarazzo

first: Gabriella

last: Minchiotti


title: Saccharomyces cerevisiae promoter library

city: Cambridge

first: Carl

middle: G

last: de Boer

state: MA

address: 415 Main St

institute: The Broad Institute

summary:
Combinatorial promoter expression level estimation via cell sorting

The purpose of this experiment was to determine the expression level of a library of synthetic promoters.  The promoters were cloned in front of a GFP reporter and the resulting library transformed into yeast, sorted by FACS into six fluorescence bins, and the contents of the bins sequenced to determine the distribution of each promoter among each fluorescence bin.  This was then used to calculate an expression level for each promoter with enough data.

overall_design:
The promoters were sorted into six bins and these, along with the unsorted library were barcoded and sequenced on a single lane of an Illumina HiSeq.

The following Series supplementary files are provided:
allPromoters.fsa.txt: the sequences of the promoters corresponding to the names in allPromoters.txt, not actually fasta format. Promoter sequence starts at pos 155 (0 indexed).
allPromoters.txt: the names of all the promoters, corresponding to the sequences in allPromoters.fsa.txt
barcodes.txt: the sequncing barcodes, corresponding to read2 from the sequencing files.

first: Harm

last: van Bakel

first: Kyle

last: Tsui

first: Joyce

last: Li

first: Quaid

middle: D

last: Morris

first: Corey

last: Nislow

first: Jack

middle: F

last: Greenblatt

first: Timothy

last: Hughes


title: Evaluation of quantitative miRNA gene expression platforms in the microRNA Quality Control (miRQC) study

city: Bangalore

first: Aishwarya

last: Narayanan

address: 5th Floor, KBP, Bellary Road

country: India

institute: Strand Life Sciences

summary: MiRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. Several measurement platforms, designed to determine their relative RNA abundance levels in biological samples, have been developed. In this study, we systematically compared 12 commercially available miRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from miRNA family members with varying homology. We developed novel and robust quality metrics to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, accuracy, specificity, and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which helps to guide informed selection of a quantitative miRNA gene expression platform in function of particular study goals.

sample_organism:
Homo sapiens
Escherichia phage MS2

platform_organism:
Homo sapiens
Escherichia phage MS2

overall_design: Sequencing of 20 miRQC samples on Illumina Genome Analyzer IIx System

first: Gary

last: Schroth

first: Vamsi

last: Veeramachaneni


title: The differences of the transcriptional profile between wide-type maize and transgenic ZmPIS maize (drought tolerance) by the assay of digital gene expression profile data

city: Jinan

first: Xiuxia

last: Liu

address: 27 Shanda South Road

institute: Shandong University

summary: We report the application of single-molecule-based sequencing technology for high-throughput profiling of transcriptome in mazie plants. The ZmPIS gene coding PtdIns synthase from maize with a maize ubiquitin promoter was transferred into maize. The transgenic ZmPIS maize showed enhanced drought tolerance compared to non-transgenic maize. The differentially expressed genes between wide-type maize and transgenic ZmPIS maize were detected by the assay of digital gene expression profile and real time RT-PCR datas. The results displayed that the overexpression of ZmPIS resulted in the expression levels changes of a large number of genes including genes involved in the phosphatidylinositol (PI) metabolic pathway, photosynthesis metabolism, carbohydrate metabolism, aminoacid metabolism and genes coding transcription factors.

overall_design: Examination of The differences of the transcriptional profile between wide-type maize and transgenic ZmPIS maize and analysis of the network regulated by the ZmPIS gene

first: Juren

last: Zhang


title: Genome and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei

city: Beijing

first: Jiang

last: Zhengqiang

address: Qinghua East Road, Haidian Distric

department: Department of Biotechnology

institute: College of Food Science and Nutritional Engineering, China Agricultural University

summary:
The zygomycete fungi-like Rhizomucor miehei have been extensively exploited for the production of various enzymes. As a thermophilic fungus, R. miehei is capable of growing at temperatures that approach the upper limits for all eukaryotes. In order to study the thermophilic mechanism, the transcriptional profiles of R. miehei CAU432 grown at two different temperatures (at 30°C and 50°C) were investigated by RNA-seq analysis.
Approximately 35 million high-quality reads were generated from each library, and 62% reads were uniquely mapped to the genome. A high percentage of reads (67.1%) were mapped to predicted protein-coding genes, while 3.96% reads were distributed in splice junctions, 3.49% reads in antisense transcripts, 2.27% in introns, and 21.9% in other genomic regions. The frequency of reads which mapped to different genes ranged from one to over 300,000. More than 90% of predicted genes (9,680, 93% at 30°C; 9,618, 93.6% at 50°C) were detected with at least one read, while 128 genes and 190 genes were uniquely expressed at 50°C and 30°C, respectively. The results show that 2,117 genes were differently expressed (P<0.001) by the fungus with more than two-fold changes. These genes include 849 up-regulated and 1,268 down-regulated genes in mycelia grown at 50°C. These significantly differently expressed genes include many genes, putatively involved in thermophilic process, such as HSP, chaperones and proteasome.

overall_design: 2 samples examined: mycelium growing at 30°C and 50°C

first: Zheng-Qiang

last: Jiang

first: Bing

first: Guo-Qiang

first: Shang-Wu

last: Chen


title: An allele-aware approach identifies altered nuclear associations of the contracted D4Z4 array at the FSHD locus

city: Seattle

last: Siebenthall

state: Washington

address: 1100 Fairview Ave N

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

summary: Facioscapulohumeral dystrophy (FSHD) is caused by a deletion in a D4Z4 macrosatellite repeat array in the 4q subtelomere that leads to somatic de-repression of the transcription factor DUX4. It is not fully understood how array contractions cause de-repression, but they alter the local chromatin structure of D4Z4, resulting in loss of heterochromatic markers. In order to determine whether pathogenic contractions also alter the nuclear organization of the FSHD locus, we designed an allele-aware, high-throughput circular chromosome conformation capture assay (4C-seq) that distinguishes the two 4q copies of the locus from each other and from a paralogous locus in the 10q subtelomere. Using a short sequence length polymorphism (SSLP) four kilobasepairs from the array as âharacterized the genomic contacts made by the four copies of the FSHD locus in muscle cell nuclei. The SSLP contacts predominate on the same chromosome arm as the bait, decaying rapidly beyond 30-40 Mb. Contacts on other chromosomes were enriched near centromeres and CTCF sites. We also found that the FSHD locus normally contacts regions adjacent to the nuclear lamina and with low overall gene expression. However, we found that the deleted locus in FSHD cells makes more contacts with regions with comparatively lower lamina adjacency and higher overall gene expression than in control cells. Furthermore, association of D4Z4 with components of the nuclear lamina was reduced in FSHD cells relative to control cells. Our results suggest that altered nuclear organization at 4q35 may be one factor in the de-repression of DUX4 in FSHD.

overall_design: 5 primary cells (2 control, 1 FSHD1, 1 FSHD2, and 1 FSHD3) were used to generate 4C libraries in biological duplicate, for a total of 10 4C libraries

middle: T

first: Janet

last: Young

first: Oliver

middle: R

last: Serang

first: Jessica

last: de Greef

first: Judit

last: Balog

first: Silvere

last: van der Maarel

first: Stephen

middle: J

last: Tapscott

first: Barbara

last: Trask


title: Transcriptional profiling of Neurospora crassa conidia undergoing germling fusion: Δpp-1 vs WT

status: Public on Aug 30 2013

city: Berkeley

first: Abby

last: Leeder

state: CA

address: 111 Koshland Hall

institute: UC Berkeley

summary: To study the consequences of transcription factor pp-1 deletion during vegetative cell fusion, we sequenced mRNA of WT and Δpp-1 conidia during a period of intense germling fusion activity .

first: Abigail

middle: C

first: Wilfried

last: Jonkers

first: Jingyi

last: Li

first: Monika

last: Fisher

first: N

middle: L

last: Glass