title: Reconstructing gene regulatory networks of tumorigenesis

first: Valeriya

last: Malysheva


title: Whole genome transcriptome analysis of Group A Streptococcus (Streptococcus pyogenes) isolates from the three distinct genetic clades of M89 population

status: Public on May 12 2016

city: Houston

first: Priyanka

last: Kachroo

address: 6670 Bertner Avenue

country: USA

institute: Houston Methodist Research Institute

type: Expression profiling by high throughput sequencing

summary: The goal was to perform global transcriptome analysis on genetically representaive pre-epidemic and epidemic M89 strains to investigate how the genetic differnces may have altered gene expression potentially contributing to epidemic emergence and persistence.

overall_design: The transcriptomes of 6 GAS emm89/M89 strains (one strain representative of each of clades 1, 2 and 3, and 3 strains from subclade3D) were compared at two phases of growth, mid-exponential and early-stationary, using 3 biological replicates, to identify differences in gene expression potentially contributing to the evolution and emergence of epidemic clade 3 and subclade 3D strains.

first: James

middle: M

last: Musser

first: Stephen

middle: B

last: Beres


title: H3K36 mutations promote sarcomagenesis through genome-wide remodeling of H3K36 and H3K27 methylation

status: Public on May 13 2016

city: Madison

first: Peter

middle: W

last: Lewis

state: Wisconsin

address: 330 N Orchard Street

department: Department of Biomolecular Chemistry

institute: University of Wisconsin-Madison

type: Genome binding/occupancy profiling by high throughput sequencing

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

sample_organism:
Drosophila melanogaster
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Circuit-wide transcriptional profiling reveals region specific gene co-expression networks regulating depression susceptibility

city: New York

first: Immanuel

last: Purushothaman

state: New York

address: 1425 Madison Avenue

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

summary: Depression is a complex and heterogeneous disorder and a leading contributor to the global burden of of disease. Most previous research has focused on individual brain regions and individual genes that contribute to depression. However, emerging evidence in both humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here we use a bioinformatics approach intersecting differential expression analysis with weighted gene co-expression network analysis to identify transcriptional networks that regulate susceptibility to depressive-like symptoms in mice. We performed RNA-sequencing on multiple brain regions from control animals and those either susceptible or resilient to chronic social defeat stress (CSDS) at multiple time points after defeat. We bioinformatically identified several transcriptional networks that regulate depression susceptibility, and in vivo manipulations of these networks confirmed their functional significance at the levels of gene transcription, synaptic regulation, and behavior. Our findings reveal novel transcriptional networks that control stress susceptibility and offer fundamentally new leads for antidepressant drug discovery.

overall_design: RNA-seq samples were generated from 4 brain regions (nucleus accumbens (NAC), prefrontal cortex (PFC), amygdala (AMY) and ventral hippocampus (VHIP) ) at 3 time-points (48h, 28d, 28d +1h stress) after chronic social defeat stress in control, susceptible and resilient mice. Additionally, RNA-seq samples were generated from virally infected VHIP tissue (HSV-GFP or HSV-Dkkl1) after an accelerated social defeat to assess the effect of Dkkl1 over-expression.

first: Rosemary

last: Bagot

first: Li

last: Shen


title: Global Mapping of Human RNA-RNA Interactions

city: Toronto

first: Timothy

last: Sterne-Weiler

state: Ontario

address: 160 College Street, Room 1030

department: The Donnelly Centre

country: Canada

institute: University of Toronto

summary: We describe a new method enabling the global-scale mapping of RNA-RNA duplexes cross-linked in vivo, ‘LIGation of interacting RNA followed by high-throughput Sequencing’ (LIGR-Seq). Applying this method in human cells reveals a remarkable landscape of new RNA-RNA interactions involving all major classes of ncRNA, and mRNA. LIGR-Seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including interactions involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-Seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.

overall_design: RNA-RNA interaction discovery in HEK293T cells in two replicates.

first: Eesha

last: Sharma

middle: H

first: Dave

last: O' Hanlon

first: Benjamin

middle: J

last: Blencowe


title: Gene expression profile of chemical-induced neural stem cells from defined fibroblasts

city: San Francisco

first: Gladstone

last: Bioinformatics

state: CA

address: 1650 Owens St

department: Data Science and Biotechnology

institute: Gladstone Institutes

summary: Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small molecule-driven approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging to develop. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine small molecules (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts towards a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study therefore provides an effective chemical approach for generating neural stem cells from mouse fibroblasts, and reveals mechanistic insights into underlying reprogramming process.

overall_design: Total RNA from mouse tdMEFs that were collected at day 0 (D0), day 4 (D4), day 8 (D8), and day 12 (D12) after M9 treatment, ciNSLCs, as well as two positive controls, including mouse primary embryonic neural progenitor cells and neural stem cell line SCR029, were used for library preparation and RNA-seq.

first: Mingliang

last: Zhang

first: Sheng

last: Ding


title: Functional characterization of RNA-binding protein IMP2 in primary Glioma cell lines

city: Basel

first: Tommy

middle: Beat

last: Schlumpf

state: Basel-Stadt

address: Mattenstrasse 26

department: D-BSSE

country: Switzerland

institute: ETH Zürich

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Gfi1 as a new predictive and therapeutical target of MDS/AML

city: Montreal

first: Möröy

last: Tarik

state: Quebec

address: 110 Pine avenue West

institute: Insititut de Recherches Cliniques

summary:
MDS is characterized by a disturbed function of the myeloid lineage of the hematopoietic system that may transform to AML, a malignant disease of the myeloid compartment. Epigenetic and genetic aberrations contribute to the initiation and progression of MDS/AML. GFI1 is a transcriptional repressor, which regulates expression of its target genes by, among other approaches, recruiting HDACs to its target genes to remove histone 3 lysine 9 (H3K9) acetylation, a marker for active gene expression. Low levels of GFI1 expression and deletion of one GFI1 allele contribute to MDS/AML development in human patients and are associated with a specific gene expression signature and inferior prognosis. To explore the mechanism behind this, we used a mouse strain, which expresses GFI1 only at 5-10% of the normal level (GFI1-Knock-down (KD)). Knock-down of GFI1 or loss of one murine Gfi1 allele reduced latency and increased incidence of AML in different murine models of human MDS/AML development. On the epigenetic level, KD of Gfi1 lead to increased amount of H3K9 acetylation, resulting in increased expression of genes involved in AML development. On a translational level both murine as well as human AML cells with low expression of GFI1 are resistant to standard epigenetic therapy. We show that treatment with histone acetyltransferase inhibitors might be a novel treatment approach for low Gfi1-expressing blast cells. GFI1 has a dose dependent role in myeloid malignancies and is a biomarker for therapeutic intervention.
We carried out ChIP-Seq Analysis of H3K9Ac and RNA-Seq in leukemic cells from mice expressing reduced levels of Gfi1 compared to controls expressing normal levels of the factor.

overall_design: Leukemic cells were extracted from 2 mice from each genotype, Gfi1-KI and Gfi1-KD. H3 and H3K9Ac ChIP-Seq as well as RNA-Seq was carried out on each sample.

first: Judith

last: Hönes

first: Lacramioara

last: Botezatu

first: Tarik

last: Möröy

first: Cyrus

last: Khandampour


title: Injury-induced enhancer remodelling in spinal microglia - a novel mechanism for pain chronification?

city: London

first: Franziska

last: Denk

address: Guy's Campus

department: Wolfson SPaRC

country: United Kingdom

institute: King's College London


title: Injury-induced enhancer remodelling in spinal microglia - a novel mechanism for pain chronification? [ChIP-Seq]

summary: Chronic pain is common and devastating. Yet, its precise molecular origins remain unclear. The condition induces well-characterised changes in neurons and microglia, but it is unknown why they persist long after the precipitating injury has healed. We posit a role for enhancers - regions of open chromatin that define a cell’s transcription factor binding profile. Enhancer profiles can alter upon environmental stimulation, functioning as a kind of molecular memory. Here, a mouse model of persistent neuropathic pain was used to examine microglial enhancers with flow cytometry and sequencing. We observed injury-specific alterations of enhancers in close proximity to transcriptionally regulated genes. Our data also shine light on details relating to the spinal cord immune response and provide the first genome-wide gene expression profile of isolated microglia in a pain state. We hypothesise that enhancer deposition may constitute a novel mechanism by which painful experiences are encoded on a molecular level.

overall_design: ChIP-seq and RNA-seq of isolated spinal cord microglia after peripheral spinal nerve ligation or sham surgery in mice (day 7)

first: F

first: M

last: Crow

first: A

last: Didangelos

first: D

last: Lopes

first: S

last: McMahon


title: Injury-induced enhancer remodelling in spinal microglia - a novel mechanism for pain chronification? [RNA-Seq]


title: MLL1 and MLL fusion proteins play distinct roles in regulating MLLleukemic transcription program

city: Ann Arbor

first: Yali

last: Dou

state: MI

address: 1301 Catherine Street

department: Pathology Department

institute: University of Michigan


title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

city: San Antonio

first: Chin-Hsing Annie

last: Lin

state: TX

address: One UTSA Circle

department: Biology

institute: University of Texas at San Antonio

summary: Neural stem progenitor cells (NSPCs) in the human subventricular zone (SVZ) potentially contribute to life-long neurogenesis, yet subtypes of glioblastoma multiforme (GBM) contain NSPC signatures that highlight the importance of cell fate regulation. Among numerous regulatory mechanisms, the post-translational methylations onto histone tails are crucial regulator of cell fate. The work presented here focuses on the role of two repressive chromatin marks tri-methylations on histone H3 lysine 27 (H3K27me3) and histone H4 lysine 20 (H4K20me3) in the adult NSPC within the SVZ. To best model healthy human NSPCs as they exist in vivo for epigenetic profiling of H3K27me3 and H4K20me3, we utilized NSPCs isolated from the adult SVZ of baboon brain (Papio Anubis) with brain structure and genomic level similar to human. The putative role of H3K27me3 in normal NSPCs predominantly falls into the regulation of gene expression, cell cycle, and differentiation, whereas H4K20me3 is involved in DNA replication/repair, metabolism, and cell cycle. Using conditional knock-out mouse models to diminish EZH2 and Suv4-20h responsible for H3K27me3 and H4K20me3, respectively, we found that both repressive marks have irrefutable function for cell cycle regulation in the NSPC population. While both EZH2/H3K27me3 and Suv4-20h/H4K20me3 have implication in cancers, our comparative genomics approach between healthy NSPCs and human GBM specimens revealed that significant sets of genes enriched with H3K27me3 and H4K20me3 in the NSPCs are altered in the human GBM. In sum, our integrated analyses across species highlight important roles of H3K27me3 and H4K20me3 in normal and disease conditions in the context of NSPC.

overall_design: SVZ H3K27me3 and H4K20me3 ChIP-seq profiles of an adult baboon brain was generated by deep sequencing with Illumina HiSeq 2000.

first: Christopher

middle: T

last: Rhodes

first: Chin-Hsing

middle: A


title: MeDIP on the marine medaka sperm (Oryzias melastigma)

status: Public on May 11 2016

city: Boston

first: Eric

last: Greer

state: MA

address: 320 Longwood Avenue

department: Newborn Medicine/Department of Pediatrics

institute: Boston Children's Hospital/Harvard Medical School

type: Methylation profiling by high throughput sequencing

summary: Hypoxia is amongst the most widespread and pressing problems in aquatic environments. For the first time, we demonstrate that fish (Oryzias melastigma) exposed to hypoxia show reproductive impairments in F2 generations despite these progenies (and their germ cells) having never been exposed to hypoxia. We further show that the observed transgenerational reproductive impairments are associated with a differential methylation pattern of specific genes in sperm of both F0 and F2.

overall_design: Examination of Methylated DNA immunoprecipitation on 4 groups (F0N, F0H, F2T, F2H)

first: Rudolf

middle: S

last: Wu


title: MLL1 and MLL fusion proteins play distinct roles in regulating MLLleukemic transcription program [RNA-Seq]

summary: This study describes the transcriptome profiling of: 1) MM-401, a MLL1 specific inhibitor in MAF9 cells; 2) Comparation of the transcriptone profilings of MM-401,EPZ-5676 (Dot1L inhibitor), MI-2(Menin inhibitor) and  iBET(Brd4 inhibitor) in MAF9 leukemia cells.

overall_design: RNA-Seq profiling on mouse MAF9 leukemia cells. Biological duplicates of each sample are labled as rep1/2.

first: Jing

last: Xu


title: Epigenetic features of differentiation-regulated replication origins

city: Bethesda

first: Christophe

middle: E

last: Redon

state: MD

address: 37 Convent Drive

institute: NIH

summary: To identify chromatin states and histone modifications that locally mark replication origins, we profiled origin distributions in eight human cell lines representing embryonic and differentiated cell types. Consistent with a role for chromatin in determining origin activity, we found that cancer and non-cancer cells of similar lineages exhibited highly similar replication origin distributions. Surprisingly, our study revealed that DNAse hypersensitivity, which often correlates with early replication at large-scale chromatin domains, did not emerge as a strong local determinant of origin activity. Instead, we found that two distinct sets of chromatin modifications exhibited strong local associations with two discrete groups of replication origins. The first origin group consisted of about 50,000 regions that actively initiated replication in all cell types examined and preferentially colocalized with unmethylated CpGs and with the euchromatin markers, H3K4me3 and H3K9Ac. The second group consisted of origins that were consistently active in cells of a single type or lineage and preferentially colocalized with the heterochromatin marker, H3K9me3. Shared origins replicated throughout the S-phase of the cell cycle, whereas cell-type specific origins preferentially replicated during late S-phase.

overall_design: Nascent strands were purified with the lambda exonuclease methods from normal and cancer cells and sequenced.

middle: I

last: Aladjem


title: Ribosome profiling study of Dhh1p overexpression and the dhh1 knockout strain using monosome-protected footprints

city: Baltimore

first: Aditya

last: Radhakrishnan

address: 725 N. Wolfe St., PCTB 704

department: Molecular Biology and Genetics

institute: Johns Hopkins School of Medicine

summary: A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. In this study we establish that the DEAD-box helicase Dhh1p is the sensor of codon optimality (i.e. translational elongation rate) that targets an mRNA for decay. First, we find that mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a DHH1-dependent manner. Biochemical experiments show that Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find that these effects on mRNA decay are sensitive to the number of slow moving ribosomes on an mRNA. Using a tethering system, we establish that non-optimal mRNAs become preferentially saturated with ribosomes when Dhh1p is bound. Moreover, over-expression of Dhh1p leads to the accumulation of ribosomes specifically on mRNAs with low codon optimality in ribosome profiling experiments. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.

overall_design: 18 biological samples are included in the study (9 ribosome footprinting samples and 9 mRNA-Seq samples). These include wild-type, Dhh1 knockout, and Dhh1 overexpression strains (with biological replicates), as well as catalytically inactive Dhh1 overexpression. Also included are ribosome footprint profiling and mRNA-Seq of overexpression mCherry reporter mRNA with catalytically active or inactive overexpressed Dhh1 tetherered.

first: Rachel

last: Green


title: Dynamic changes in E protein activity orchestrate germinal center and plasma cell development

city: Clayton

first: Wei

last: Shi

state: VIC

address: 15 Innovation Walk

department: Department of Biochemistry and Molecular Biology

country: Australia

institute: Monash University


title: Arabidopsis AtMORC4 and AtMORC7 form Nuclear Bodies and Repress a Large Number of Protein-Coding Genes

city: Cambridge

first: C. Jake

last: Harris

address: Downing Street

department: Department of Plant Sciences

institute: University of Cambridge

summary: The MORC family of GHKL ATPases are an enigmatic class of proteins with diverse chromatin related functions. In Arabidopsis, AtMORC1, AtMORC2, and AtMORC6 act together in heterodimeric complexes to mediate transcriptional silencing of methylated DNA elements. Here, we studied Arabidopsis AtMORC4 and AtMORC7. We found that, in contrast to AtMORC1,2,6, they act to suppress a wide set of non-methylated protein-coding genes that are enriched for those involved in pathogen response. Furthermore, atmorc4 atmorc7 double mutants show a pathogen response phenotype. We found that AtMORC4 and AtMORC7 form homomeric complexes in vivo and are concentrated in discrete nuclear bodies adjacent to chromocenters. Analysis of an atmorc1,2,4,5,6,7 hextuple mutant demonstrates that transcriptional de-repression is largely uncoupled from changes in DNA methylation in plants devoid of MORC function. However, we also uncover a requirement for MORC in both DNA methylation and silencing at a small but distinct subset of RNA-directed DNA methylation target loci. These regions are characterized by poised transcriptional potential and a low density of sites for symmetric cytosine methylation. These results provide insights into the biological function of MORC proteins in higher eukaryotes.

overall_design: This dataset includes: 6 BS-seq and 26 RNA-seq datasets

first: C


title: Functional characterization of RNA-binding protein IMP2 in primary Glioma cell lines [HTS]

first: Tommy

last: Schlumpf

summary: Cancer stem cells (CSC) dictate tumor cell heterogeneity in diverse cancer types and arise, in part, from microRNA (miRNA)-dependent alteration of gene expression. The let-7 miRNA family induces differentiation by silencing genes that maintain stemness and is repressed by the RNA-binding proteins LIN28A/B, which preserve stemness in normal embryonic and malignant cells. Here, we observed that LIN28A/B is undetectable in glioma stem cells (GSC) whereas let-7 and, paradoxically, their target genes are highly expressed. Using photoactivatable-ribonucloside-enchanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor-2 mRNA-binding protein 2 (IMP2) protects let-7 target genes from silencing and provides a mechanistically distinct alternative to LIN28A/B toward both GSC and neural stem cell specification. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism by which it supports GSC maintenance.

overall_design: We characterized RNA binding sites of IMP2 in MGG8 Glioma cancer stem cells and differentiated cells. To profile changes upon IMP2 modulation we performed RNA-Seq in MGG4 and 8 cells in both states upon IMP2 knockdown and overexpression. Furthermore we investigated changes during differentiation from cancer stem cells to adherent cells in 6 and 7 RNA-Seq datasets in MGG4 and 8 cells.

first: Nils

last: Degrauwe

first: Renato

last: Paro

first: Ivan

last: Stamenkovic


title: MLL1 and MLL fusion proteins play distinct roles in regulating MLLleukemic transcription program [ChIP-Seq]

first: Yali

summary: This study describes the epigenetic profiling change of 1.) Mll1,  WDR5 and H3K4me2 in mouse MAF9 leukemia cells treated with or without MM401; 2.) WDR5 and H3K4me2 epigenetic profiling in MLL1 flox/flox, ER-Cre+/- cells with knocking out MLL1 by 4OHT or not.

overall_design: ChIP-Seq profile of Mll1, WDR5 and H3K4me2 were generated in MAF9 cells or MLL1 flox/flox, ER-cre+/- cells. A sequence profile of genomic DNA (Input) is also included.


title: Negative control of CSL gene transcription by stress/DNA damage response and p53 [RNA-Seq]

city: Epalinges

first: Gian-Paolo

last: Dotto

address: Chemin de Boveresses, 155

department: Biochemistry

institute: UNIL

summary: CSL is a key transcription factor, mostly acting as a repressor. While known as main effector of Notch signaling, it can also play Notch-independent functions. Despite the wide interest in CSL, the mechanisms responsible for its own regulation have been little studied. We recently showed that CSL down-modulation in human dermal fibroblasts (HDFs) leads to conversion into cancer associated fibroblasts, which promote keratinocyte tumor development. We show here that levels of CSL gene transcription differ among HDF strains derived from many different individuals, with negative correlation with genes involved in DNA damage/repair. CSL expression in all tested strains is negatively regulated by stress / DNA damaging insults caused by UVA, Reactive Oxygen Species (ROS), smoke extract and doxorubicin treatment. p53, a key effector of the DNA damage response, functions as common negative regulator of CSL gene transcription, through both suppression of CSL promoter activity and, indirectly, through increased p21 expression. CSL was previously shown to bind p53 suppressing its activity. The present findings indicate that p53, in turn, decreases CSL expression, which can serve to enhance p53 activity in the acute response of cells to DNA damaging cancer-threatening conditions.

overall_design: RNA sequencing of 46 human foreskin fibroblasts

first: Elena

last: Menietti

first: Xiaoying

first: Paola

last: Ostano

first: Jean-Marc

last: Joseph

first: Karine

last: Lefort

first: G

middle: P


title: The genomic landscape of PUF binding in stem cells

city: Stanford

first: Douglas

last: Porter

address: 269 Campus Drive

department: Dermatology

institute: Stanford

summary: We determined the genomic landscape of FBF-1 and FBF-2 binding in germline stem cells using iCLIP, a method that allows identification of protein-RNA interactions at high resolution. We first developed reagents to explore the genomic binding landscapes of full-length FBF-1 and FBF-2 in vivo and then used our iCLIP data to test the precision of several commonly used methods for CLIP peak calling. Based on this iCLIP data, we discovered that FBF-1 and FBF-2 have similar global protein-RNA interaction profiles and that they both target conserved cell cycle regulators and lincRNAs. We found that FBF-1 and FBF-2 regulate their targets through canonical as well as unexpected motif sequences. We elucidated the first in vivo crosslink site analysis for a PUF protein from which we precisely determined FBF-1 and FBF-2 binding sites. Taken together, our data provide an updated model of PUF binding in stem cells. Our study also provides new insight on the control of gene expression in stem cells by RNA binding proteins.

overall_design: iCLIP was performed on FLAG-tagged FBF-1 and FBF-2 integrated as a single copy into the C. elegans genome. Wild type worms (N2) went through identical protocols as a negative control.

first: Aman

last: Prasad

middle: F

first: Marvin

last: Wickens

first: Judith

last: Kimble


title: Negative control of CSL gene transcription by stress/DNA damage response and p53


title: Negative control of CSL gene transcription by stress/DNA damage response and p53 [ChIP-Seq]

overall_design: ChIP sequencing of DMSO vs Nutlin-3a treated foreskin HDF

last: Xu


title: MicroRNAs of Toxocara canis and their predicted functional roles.

city: Rongchang

first: Guangxu

last: Ma

state: Chongqing

address: xueyuan road 160#

department: veterinary medicine

country: China

institute: Southwest university

type: Non-coding RNA profiling by high throughput sequencing

summary: To encourage biological and biotechnological investigations on Toxocara canis, an important neglected tropical parasite, high-throughput sequencing was carried out to generate the sRNAs resource of adult Toxocara canis. In total, 11,632,676 and 10,723,433 sRNAs reads were yielded from male and female libraries, with similar proportion of small nucleolar RNA, micro RNA and small nuclear RNA. No PIWI-associated RNA was found in T. canis. 1,985 of male and 2,062 of female known miRNAs were respectively obtained, as well as 99 and 71 novel miRNAs in both genders. Comparative analysis was carried out on expression level which generated the lists of differential-expressed miRNAs, including 854 of male-specific expressed and 932 of female-specific expressed MiRNAs, and on sequence level which showed expression-sequence coordination. Based on Gene Ontology annotation and KEGG pathway analysis, the conservation and functional diversification of these differentially expressed miRNAs were investigated focusing on reproduction and larval development. 53 miRNA seed family, such as Tc-miR-1648/6090/7412/7931 and Tc-miR-698/1892/6795/6963/6980/7044/7078, were predicted to be involved in reproduction and development processes. Moreover, it was found that seed family with interspecies conservation, like ACCCGUA/ACCCUGU/CCCUGAG/GAAAGAC/GAGAUCA, have wide distribution in larvae, secretion and host serum, strongly suggesting the potential roles of these miRNAs in host and parasite interactions. This is the first exploration of sRNAs in T. canis, which would boost systematic biological investigations and encourage novel drugs vaccines and diagnostic tests.

overall_design: High-throughput small RNA sequencing in male and female adult Toxocara canis.

first: Rongqiong

last: Zhou


title: Synchronized translation programs across cellular compartments

first: Mary

last: Couvillion

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Oxidative phosphorylation (OXPHOS) is fundamental for life. OXPHOS complexes pose a unique challenge for the cell, because their subunits are encoded on two different genomes, the nuclear genome and the mitochondrial genome. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to the mitochondrial system, thus the co-regulation of OXPHOS genes remains largely unexplored. Here we globally monitored mitochondrial and nuclear gene expression processes during mitochondrial biogenesis when OXPHOS complexes are synthesized. Nuclear- and mitochondrial- encoded OXPHOS transcript levels do not increase concordantly. Instead, we observe that mitochondrial and cytosolic translation are rapidly and dynamically regulated in a strikingly synchronous fashion. Furthermore, the coordinated translation programs are controlled unidirectionally through the intricate and dynamic control of cytosolic translation. Thus the nuclear genome carefully directs the coordination of mitochondrial and cytosolic translation to orchestrate the timely synthesis of each OXPHOS complex, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for global gene regulatory studies of mitochondrial biology.

overall_design: Examination of whole-cell RNA levels (rRNA-depleted RNA-seq) and both cytosolic and mitochondrial translation regulation (cytoribosome profiling and mitoribosome profiling) through mitochondrial biogenesis in S. cerevisiae

first: Iliana

middle: C

last: Soto

first: Gergana

last: Shipkovenska

first: Stirling

middle: L

last: Churchman


title: Analysis of Hybrid Incompatability between C nigoni and C briggsae

first: Peter

last: Sarkies

address: Du Cane Road

department: MRC Clinical Sciences Centre

institute: Imperial College London

summary: An introgression of C. briggsae onto the X chromosome of C briggsae results in male sterility.  In order to determine the cause, we sequenced small RNAs from this strain and compared to fertile males of the two parent species.

sample_organism:
Caenorhabditis briggsae
Caenorhabditis nigoni

overall_design: Small RNA sequencing was carried out from 200 adult males of each species

first: Zhongying

last: Zhao


title: EZH1/SUZ12 complex positively regulates the transcription of NF-κB target genes via interaction with UXT

first: Pin-Ji

last: Lei

state: Massachusetts

address: 100 Blossom Street

department: Department of Radiation Oncology

institute: Massachusetts General Hospital

summary: EZH1 is one of polycomb group genes which positively regulates transcription in the genome-wide scale. However, the underlying mechanisms still remain elusive. Here we report that EZH1 physically interacts with UXT, one small chaperon-like transcription co-activator of NF-κB. UXT specifically interacts with EZH1 and SUZ12, but not EED or EZH2. Similar to UXT, RNA interference of EZH1 and SUZ12 in the cell impairs the transcriptional activation of NF-κB target genes after TNFα treatment, and increases the induced cell death. Then RNA-Sequencing analysis was used to analysis the transcriptome in HCT116 cells after EZH1 or UXT knockdown. EZH1 or UXT deficiency nearly impaired the induced expression of all the genes by TNFα. ChIP-Sequencing was used to analysis the H3K27me1, H3K27me2 and H3K27me3 levels in HCT116 cells after EZH1 or UXT knockdown.  However, the results show that H3K27 mono-, di- and trimethylation on NF-κB target genes are not affected with EZH1 or UXT deficiency. EZH1 does not affect the translocation of RELA/p65 from cytosol to nuclear either. Instead, EZH1 regulates the recruitment of RELA/p65 and RNA POLII to target genes. Taken together, our study demonstrates EZH1 as a positive regulator for NF-κB signaling and reveals the underlying mechanism how EZH1 and UXT work together in transcription regulation.

overall_design: RNA-Sequencing and ChIP-Sequencing were used to analysis the transcriptome and H3K27 methylations in HCT116 cells after EZH1 or UXT knockdown.

first: Shuai-Kun

last: Su

first: Chun-Yuan

last: Li


title: Next-Generation Sequencing reveals the effects of MYC silencing in gastric cancer cell lines from northern Brazil

status: Public on May 10 2016

city: Rio de Janeiro

first: Daniel

last: Carvalho

state: Rio de Janeiro

address: Caixa Postal 26820

country: Brazil

institute: GenOne Biotetechnologies

summary:
Purpose: In carcinogenesis, the stomach, the MYC gene is frequently amplified and its protein is overexpressed, which contributes to uncontrolled cell proliferation mucosal gástrica.Três cell lines were subjected to sequencing semiconductor Proton Ion before and after the silencing of MYC. These lines were originating from diffuse and intestinal histologic types and the third of a metastasis from a stomach tumor originating in a ascites.
Methods: mRNA profiles were generated from three established lines of CG previously characterized by our group. AGP01 (Obtained from ascitic fluid of an intestinal-type GC), ACP02 (diffuse-type GC) and ACP03 (intestinal-type GC). The three cell lines present chromosome 8 trisomy and MYC amplification, reflecting the genetic characteristics found in gastric cancer samples from Northern Brazil. They were generated by sequencing depth in triplicate using the Sequencer ™ Ion Proton. Raw data reads Obtained by primary sequencing Were Submitted to quality control in order to calculate alignment and to assess how the reads behave When Compared to the reference human genome (Hg19 / GRCh37). The aligned reads Were mapped and quantified using TMAP. Gene expression levels Were quantified using Sailfish pack software and the RPKM (Reads Per kilobase per Million mapped reads) method was used to estimate Then expression levels gene.
Results: Using analysis of an optimized workflow, we mapped more than 13 million readings sequences between our samples from the human genome. Were mapped a total of 917 up-regulated genes and 1,566 different genes down-regulated Were When comparing the expression of AGP01 non-targeting control (1C) versus AGP01 MYC-silenced (1M), with a total of 2,483 DEGS. The evaluation of ACP02 cells treated with non-targeting control (2C) versus ACP02 MYC-silenced (2M) Showed a total of 5,327 DEGS, with 1,299 up-regulated and 4,098 down-regulated genes. The total of 4,117 were found DEGS When comparing ACP03 control cells (3C) with ACP03 MYC-silenced cells, with 3,274 up-regulated and 843 down-regulated genes. Silencing of the MYC gene, allowed the detection of more than 1,000 DEGS (Differentially Expressed Genes) between the three strains that were considered significant if the False Discovery Rate (FDR) was ≤0.001, P-value <0.05, and the absolute value of the Fold change (FC) was ≥1. The analysis of DEGS showed 22% and 34% were detected with a profile down-regulated in intestinal and diffuse type, respectively. The up-regulated profile was higher in the intestinal type, with 77% of DEGS than the diffuse type with 13% DEGS. Furthermore, the lineage from metastasis showed 33% of DEGS up-regulated and 66% of DEGS down-regulated.
Conclusions: Our study is the first to describe bioinformatics methods the effect before and after the silencing of the MYC gene in cell lines of gastric cancer northern people of Brazil. Our results showed that depending on the histological type there are many functional genes between the three strains that were recorded significantly associated with pathways enriched for cancer, metabolism, adhesion, cell cycle, proteolysis and signaling, suggesting the existence of routes of different carcinogenesis that lead normal mucosa to turn into gastric cancer.

overall_design: It was help silencing MYC in three lines of gastric cancer, originating from diffuse and intestinal histologic types and the third of a metastasis from a stomach tumor originating in a ascites. These three cell lines were subjected to sequencing semiconductor Pronton Ion before and after the silencing of MYC.

first: Rommel

middle: R

last: Burbano

first: Maués

last: Jersey

first: Moreira-Nunes

last: Caroline

first: Ribeiro

last: Hellen


title: Global Regulatory Network Controlling Human Somatic Cell Reprogramming [SMAD3 and SFRS11 Knockdown]

city: Singapore

first: Chadi

last: El Farran

address: 61 Biopolis Drive

country: Singapore

institute: Institute of Molecular and Cell Biology

summary: This is the first study deciphering the global regulatory network that drives human somatic cells during epigenetic rewiring towards the pluripotent state.

overall_design: Examination of the Transcriptomic profiles of cells undergoing reprogramming.

first: Cheng-Xu

middle: D

last: Toh

last: EL Farran

first: Zheng-Shan

last: Chong

first: Julien

last: Maury

first: James

last: Collins

first: George

middle: Q

last: Daley

first: Hu

first: Yoon-Pin

last: Lim

first: Yuin-Han

last: Loh


title: Nfib promotes Metastasis through a Widespread Increase in Chromatin Accessibility

first: William

last: Greenleaf

address: 279 Campus Drive

institute: Stanford University

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

overall_design: Refer to individual Series


title: DNA methylome analyses implicate fallopian tube as the tissue of origin for high grade serous ovarian cancer

city: Omaha

first: Adam

middle: R.

last: Karpf

state: Nebraska

address: 985950 Nebraska Medical Center

department: Eppley Institute for Research in Cancer

institute: University of Nebraska Medical Center

type: Methylation profiling by genome tiling array


title: Ileal pouch transcriptomics reveal shared pathogenesis between pouchitis and ulcerative colitis

city: Charlottesville

first: Yong

last: Huang

state: VA

address: 1340 Jefferson Park Ave

department: Medicine

institute: University of Viginia

summary:
UC pouchitis is a potential model of UC. We prospectively examined the pouch transcriptomes of UC and familial adenomatous polyposis (FAP) IPAA patients to unveil molecular mechanisms of UC pouchitis susceptibility.
Methods: Total RNA was isolated using the AllPrep DNA/RNA Mini Kit (QIAGEN, Cat No. 8020). RNA quality was evaluated using Bioanalyzer (Agilent, Santa Clara, CA). All RNA samples displayed RNA Integrity Number (RIN) >7. RNAseq including cDNA library preparation was processed at the Genomics Core Facility of University of Chicago (https://fgf.uchicago.edu/). Total RNA in the amount of 100-500μg per sample was depleted of ribosomal RNA using the Ribo-Zero kit (Epicentre, Madison, WI). The directional (first strand) cDNA libraries were prepared following the guide of TruSeq Stranded Total RNA Sample Preparation kit.
Results: Unlike FAP patients, UC subjects exhibited a large set of differentially expressed genes (DEGs) between pouch and pre-pouch mucosa as early as 4 months after pouch functionalization. Functional pathway analysis of DEGs in UC pouch revealed: (1) Gain of colon-associated gene expressions and loss of ileum associated gene expressions, (2) enhanced state of immune/inflammatory response, and (3) suppressed xenobiotic, lipid, and bile acid metabolic pathways. These changes were corroborated upon reanalysis of a published larger cross-sectional study of UC and FAP patients. Moreover, >70% of DEGs mapped to published IBD and normal colonic microarray datasets displayed directional changes consistent with active UC, but not Crohn's disease.
Conclusions: UC patients exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease. The transcriptome alterations provide insights into pouchitis

overall_design: Seventeen patients with UC and four patients with FAP were recruited at the University of Chicago and the Mayo Clinic Rochester. All patients underwent a total proctocolectomy with ileal pouch anal anastomosis (IPAA) as a standard of care. UC patients underwent a pouchoscopy for biopsy of the pre-pouch ileum and pouch at 4 months, 8 months, and 12 months after ileostomy closure. None of these patients had pouchitis.

first: Sushila

last: Dalal

first: Eugene

middle: B

last: Chang


title: Transcriptome of two S. cerevisiae strain in competition in natural must

city: Paterna

first: Roberto

last: Pérez

state: Valencia

address: Av Agustín Escardino 7

country: Spain

institute: Centro Superior de Investigaciones Científicas

summary: We report RNAseq analysis of two strains in competition

overall_design: Two strains, dominant and non-dominant were sampled at different time points during a natural must fermentation, separated by flow cytometry and transcriptome was analized by RNAseq

last: Pérez-Torrado

first: Kalliopi

last: Rantsiou

first: Benedeta

last: Perrone

first: Amparo

last: Querol

first: Elisabeth

last: Navarro-Tapia

first: Luca

last: Cocolin


title: DNA methylome analyses implicate fallopian tube as the tissue of origin for high grade serous ovarian cancer [seq]

summary:
The cell and tissue of origin of high grade serous ovarian cancer (HGSC) is controversial.  While the disease was traditionally assumed to arise from the ovarian surface epithelium (OSE), recent work supports an alternative model implicating the fallopian tube fimbriae epithelium (FTE).  We used comparative DNA methylome analyses as a means to test these two competing models, hypothesizing that the HGSC methylome should more closely resemble its tissue of origin.  Using two separate analysis methods, analysis within distinct genomic contexts, and validation studies using large-scale publically available HGSC methylome data, we consistently observed that the DNA methylome of HGSC more closely resembles FTE than OSE.   These data support the FTE origin model, and suggest DNA methylome analysis as a useful approach to examine cell/tissue lineage origin in cancer.
HGSC is the most common and lethal form of epithelial ovarian cancer (EOC).  Two distinct tissues have been hypothesized as the tissue of origin of HGSC: OSE and FTE.  We hypothesized that the DNA methylome of HGSC would more closely resemble the methylome of its tissue of origin. To test this, we profiled HGSC (n=10), OSE (n=7), and FTE (n=7) primary fresh-frozen tissues, and analyzed DNA methylome using Illumina 450K arrays (n=24) and Agilent Sure Select methyl-seq (n=7).  We compared methylomes using statistical analyses of differentially methylated CpG sites (DMC) and differentially methylated regions (DMR), and methylation within a variety of different genomic contexts, including CpG island shores.  We also analyzed methylation specifically at Homeobox (HOX) genes, due to their role in tissue specification.  We used publically available HGSC methylome data (n=628) to provide additional comparison with FTE and OSE and potential validation.  This analysis revealed that HGSC and FTE methylomes were significantly and consistently more highly conserved than were HGSC and OSE.   Pearson correlations and hierarchal clustering of genes, promoters, CpG islands, CpG island shores, and HOX genes all revealed the increased relatedness of HGSC and FTE methylomes.  In addition, two different large data sets of publically-available HGSC methylome data confirmed these relationships.   In summary, our findings support the FTE origin model for HGSC, the most common and deadly EOC subtype.  Due to its tissue-specificity and biochemical stability, interrogation of the DNA methylome appears to be a valuable approach to examine cell/tissue lineage in cancer.

overall_design: Obtained fresh-frozen high grade serous cancer (HGSC) samples (n=10) from patients undergoing surgical resection at Roswell Park Cancer Institute (RPCI) and fresh-frozen patient matched normal ovarian surface epithelial (OSE) cells (n=5) and fallopian tube epithelial (FTE) cells (n=5) from patients without malignancy. Used mechanical scraping and processing of the epithelial layer of resected ovaries and fimbriae fallopian tubes immediately after removal in the operating theater.   All samples were collected using IRB-approved protocols at RPCI. Isolated genomic DNA using the Puregene Tissue Kit (Qiagen), which includes RNAse treatment.  To eliminate potentially confounding effects due to methylation phenotype, we selected HGSC samples showing similar global methylation as FTE and OSE, as determined by LINE-1 pyrosequencing. Genomic DNA (1000ng) was bisulfite converted using EZ DNA Methylation-Direct kit (Zimo Research) following the manufacturer's protocol. Samples were hybridized to Infinium Human Methylation 450K BeadChips (Illumina) according to the manufacturer's protocol. Arrays were scanned on Iscan (Illumina).


title: FoxO1 Deacetylation Decreases Fatty Acid Oxidation in beta-cells and Sustains Insulin Secretion in Diabetes

city: New York

first: Youngjung

middle: Rachel

last: Kim

state: NY

address: 1150 St. Nicholas Ave

institute: Columbia University

summary: Pancreatic beta-cell dysfunction contributes to onset and progression of type 2 diabetes. In this state beta-cells become metabolically inflexible, losing the ability to select between carbohydrates and lipids as substrates for mitochondrial oxidation. These changes lead to beta-cell dedifferentiation. We have proposed that FoxO proteins are activated through deacetylation-dependent nuclear translocation to forestall the progression of these abnormalities. However, how deacetylated FoxO exert their actions remains unclear. To address this question, we analyzed islet function in mice homozygous for knock-in alleles encoding deacetylated FoxO1 (6KR). Islets expressing 6KR mutant FoxO1 have enhanced insulin secretion in vivo and ex vivo, and decreased fatty acid oxidation ex vivo. Remarkably, the gene expression signature associated with FoxO1 deacetylation differs from wild-type by only ~2% of the > 4,000 genes regulated in response to re-feeding. But this narrow swath includes key genes required for beta-cell identity, lipid metabolism, and mitochondrial fatty acid and solute transport. The data support the notion that deacetylated FoxO1 protects beta-cell function by limiting mitochondrial lipid utilization, and raise the possibility that inhibition of fatty acid oxidation in β-cells is beneficial to diabetes treatment.

overall_design: Examined 2 different feeding state and 2 different genotypes


title: Nfib promotes Metastasis through a Widespread Increase in Chromatin Accessibility [ChIP-seq]

summary: We describe Nfib as an important regulator of chromatin accessibility in SCLC.

overall_design: We performed ChIP seq for Nfib in three different cell lines. Replicates of input and pulldowns for three cell lines is given.

first: Sarah

last: Denny

first: Dian

last: Yang

first: Julian

last: Sage

first: Monte

last: Winslow


title: Microarray analysis of splenic CD11b+Gr-1+ cells

status: Public on May 09 2016

city: Tokyo

first: Makoto

last: Ishii

address: 35 Shinanomachi Shinjyuku

department: Division of Pulmonary Medicine Department of Medicine

country: Japan

institute: Keio University School of Medicine

summary:
We extracted RNA of sorted splenic CD11b+Gr-1+ cells and performed microarray analyses. Total RNA was isolated using the manufacturer’s recommended protocol for RNeasy (Qiagen, Valencia, CA). Isolated RNA was quantified, and quality was assessed using an Agilent A 2100 BioAnalyzer with the RNA NanoChip (Agilent, Andover, MA). We used 2μg of total RNA to make single-stranded antisense cDNA with the NuGEN Technologies (San Carlo, CA) Ovation Biotin System in accordance with the manufacturer’s directions. Labeled targets were hybridized to Affymetrix (Santa Clara, CA) MOE430 2.0 GeneChip microarrays for 16h at 45°C. The arrays were washed and scanned according to Affymetrix standard protocols.
We used microarrays to detail the global gene expression of CD11b+Gr-1+ cells from the control mice and the clarithromycin-treated mice, and identified distinct classes of up-regulated genes during this process.

overall_design: Splenic CD11b+Gr-1+ cells from the control mice and the clarithromycin-treated mice were sortd, RNA extraction from these cells was hybridized on Affymetrix microarrays.

first: Ho

last: Namkoong


title: Alterations to chromatin in intestinal macrophages link IL-10 deficiency to inappropriate inflammatory responses

city: Chapel Hill

first: Shehzad

last: Sheikh

state: NC

address: 5023A Genetic Medicine Building

institute: University of North Carolina - Chapel Hill

summary: Intestinal macrophages (IMs) are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL-10 maintains the macrophage anti-inflammatory response such that loss of IL-10 results in chronic intestinal inflammation. To investigate how IL-10-deficiency alters IM programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone-marrow, from both wild-type and IL-10-deficient (Il10-/- ) mice. We identified chromatin accessibility changes associated with bacterial exposure and IL-10-deficiency in both bone-marrow-derived macrophages (BMMs) and IMs. Surprisingly, Il10-/- IMs adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, when recombinant IL-10 was added to Il10-/- cells, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL-10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model in which IL-10-deficiency leads to chromatin alterations that contribute to a loss of IM tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation.

overall_design: Investigation of chromatin accessibility and gene expression changes in response to bacteria, and how these responses are altered in the absence of IL-10

first: Jeremy

middle: M

last: Simon

first: Terrence

last: Furey

middle: Z


title: Nfib promotes Metastasis through a Widespread Increase in Chromatin Accessibility [ATAC-seq]

first: William

last: Greenleaf

summary: We describe Nfib as an important regulator of chromatin accessibility in Small cell lung cancer (SCLC).

overall_design:
ATAC seq was performed on [1] ex vivo samples from primary tumors and metastases of a mouse model for SCLC (4 samples of each plus technical replicates of 7 samples for a total of 15 samples)
[2] cell lines derived from mouse models for SCLC.
[3] Nfib loss of function in murine SCLC cell lines (shLuc are controls, sh2 is shRNA that knocks down Nfib)
[4] Nfib gain of function in murine SCLC cell lines and human SCLC lines (empty indicates control cells, Nfib indicates cells overexpressing Nfib)


title: Genome-wide map of DNA methylation to unravel the function of EBF1 C-terminus domain

city: Freiburg

first: Senthilkumar

last: Ramamoorthy

address: Stübeweg 51

department: Cellular & Molecular Immunology

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: To study the function of the C-terminus domain, DNA methylation status was profiled in Empty, Ebf1wt and Ebf1DC transduced Ebf1-/- progenitor cells.

overall_design: Freshly prepared Ebf1−/− c-Kit+ fetal liver cells were transduced with  pmys-Empty-IRES-GFP, pmys-EBF1wt-IRES-GFP or pmys-EBF1ΔC-IRES-GFP. EBF1wt- or EBF1ΔC-transduced cells were sorted as early pro-B cells (GFP+CD19+BP1-). Vector-transduced cells were sorted as only GFP+.

first: Sören

last: Boller

first: Lukas

last: Burger

first: Rabih

last: Murr

first: Dirk

last: Schübeler

first: Rudolf

last: Grosschedl


title: Genome-wide map of EBF1, H3K4me2 and H3K27me3 marks to unravel the EBF1 C-terminus domain function

summary: To study the function of C-terminus domain, EBF1, H3K4me2 and H3K27me3 ChIP was performed

overall_design: Freshly prepared Ebf1−/− c-Kit+ fetal liver cells were transduced with  pmys-Empty-IRES-GFP, pmys-EBF1wt-IRES-GFP and pmys-EBF1DC-IRES-GFP. EBF1wt- or EBF1ΔC-transduced cells were sorted as pro-B cells (GFP+CD19+). vector-transduced cells were sorted as only GFP+.


title: Whole transcriptome analysis of UUO mouse model of renal fibrosis reveals new molecular players in kidney diseases

city: Athens

first: Aristidis

last: Charonis

state: Attiki

address: Soranou Efesiou 4

country: Greece

institute: Biomedical Research Foundation of the Academy of Athens

summary: The study demontrates differences in the transcriptome ( both of protein coding transcripts and long non-coding RNAs) in the unilateral ureteric obstruction model of renal fibrosis.

overall_design: Renal tissue was studied from animals undergoing sham operation (as controls) or right ureteric ligation. Animals were sacrificed 2 and 8 days following ligation and the right kidney tissue was examined.

first: Eleni

last: Arvaniti

first: Panagiotis

last: Moulos

first: Athina

last: Vakrakou

first: Christos

last: Chatziantoniou

last: Chadjichristos

last: Kavvadas

middle: K

last: Politis


title: BACH2 regulates CD8+ T cell differentiation by controlling access of AP-1 factors to enhancers

city: Bethesda

first: Peng

last: Li

address: 9000 Rockville Pike

department: NHLBI

institute: NIH

summary: T cell antigen receptor (TCR) signaling drives distinct responses depending upon the differentiation state and context of CD8+ T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity following viral infection. BACH2 was recruited to enhancers where it limited expression of TCR-driven genes by attenuating the availability of activator protein 1 (AP-1) sites to Jun family signal-dependent TFs. In naïve cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

overall_design: RNA-Seq expression profiles of WT and Bach2-deficient CD8+ T cells isolated ex vivo by FACS sorting or harvested following in vitro stimulation with plate-bound anti-CD3 and anti-CD28 antibodies. Genome-wide ChIP-Seq measurements of BACH2, JunD, H3K4me1, H3K27ac, p300 distribution in WT or Bach2-deficient cells. Genome-wide assay for transposase accessible chromatin (ATACSeq) measurements of d5 in vitro activated CD8+ T cells.

first: Rahul

last: Roychoudhuri

first: David

last: Clever

first: Yoshiyuki

last: Wakabayashi

first: Kylie

last: Quinn

first: Christopher

middle: A

last: Klebanoff

first: Yun

last: Ji

first: Madhusudhanan

last: Sukumar

first: Robert

last: Eil

first: Zhiya

last: Yu

first: Rosanne

last: Spolski

first: Douglas

last: Palmer

first: Jenny

middle: H

last: Pan

first: Shashank

middle: J

last: Patel

first: Derek

last: Macallan

first: Giulia

last: Fabozzi

first: Han-Yu

last: Shih

first: Yuka

last: Kanno

first: Akihiko

last: Muto

first: Jun

last: Zhu

first: Luca

last: Gattinoni

first: John

last: O’Shea

first: Klaus

last: Okkenhaug

first: Kazuhiko

last: Igarashi

first: Warren

last: Leonard

first: Nicholas

last: Restifo


title: Comparison of Bach2-tdRFP high expression and low expression NP-specific IgG1 light zone GC B cells

city: Suita

first: TOMOHIRO

last: KUROSAKI

state: Osaka

address: 3-1 Yamada-oka

institute: Osaka University Immunology Frontier Research Center

summary: Despite the importance of memory B cells for protection from recurrent infection, how these cells are selected during germinal center (GC) reactions remains unclear. We show here that light zone (LZ) GC B cells with lower affinity BCRs express a less CD40 signature and relatively high levels of Bach2, being prone to enter the memory B cell pool. We also find that Bach2 contributes to memory B cell generation in a Blimp-1-independent manner and that its higher expression confers on LZ GC cells a more advantage for entering the memory B cell compartment. Thus, our data support an instructive model in which weak T cell help keep Bach2 expression relatively high, thereby being predisposed to enter the memory pool.

overall_design: mRNA expression profiles of Bach2-tdRFP low and high expression NP-specific IgG1 light zone GC B cells were generated by deep sequencing using Illumina HiSeq 1500.

first: Tomohiro

last: Kurosaki

first: Ryo

last: Shinnakasu


title: Comparison of NP specific high and low affinity IgG1 Light zone GC B cells

summary: Despite the importance of memory B cells for protection from recurrent infection, how these cells are selected during germinal center (GC) reactions remains unclear. We show here that light zone (LZ) GC B cells with lower affinity BCRs express a less CD40 signature and relatively high levels of Bach2, being prone to enter the memory B cell pool. We also find that Bach2 contributes to memory B cell generation in a Blimp-1 independent manner and that its higher expression confers on LZ GC cells a more advantage for entering the memory B cell compartment. Thus, our data support an instructive model in which weak T cell help keeps Bach2 expression relatively high, thereby being predisposed to enter the memory pool.

overall_design: mRNA expression profiles of NP specific high and low affinity IgG1 LZ GC B cells were generated by deep sequencing using Illumina HiSeq 1500


title: High throughput profiling of microRNAs (miRNAs) in HCV-infected hepatocyte-like cells

city: Strasbourg

first: Mirjam

middle: B.

last: Zeisel

address: 3, rue Koeberlé

country: France

institute: Inserm

summary: Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in cellular homeostasis within the liver and deregulation of the miRNome has been associated with liver disease including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross-talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here, we combined a hepatocyte-like based model system, high-throughput small RNA-sequencing, computational analysis and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least two-fold including miRNAs that were previously described to target genes associated with inflammation, fibrosis and cancer development. Further investigation demonstrated that miR-146a-5p was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes as well as in liver tissues from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p over-expression is related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p positively impacts on late steps of the viral replication cycle thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease.

overall_design: Small RNA-Seq was performed in hepatocyte-like Huh7.5.1dif cells that were subjected to 7 days infection using HCVcc Jc1E2FLAG (three samples). Uninfected cells were analyzed in parallel as a control (three samples).

first: Simonetta

last: Bandiera

first: Sophie

last: Pernot

first: Hussein

last: El Saghire

first: Sarah

last: Durand

first: Christine

last: Thumann

first: Emile

last: Crouchet

first: Tao

last: Ye

first: Isabel

last: Fofana

first: Jochen

last: Barths

first: Catherine

last: Schuster

first: Patrick

last: Pessaux

first: Markus

last: Heim

first: Thomas

middle: E

last: Baumert


title: HCV-induced up-regulation of miR-146a-5p in hepatocytes promotes viral infection and metabolic pathways associated with liver disease pathogenesis


title: Genome-wide DHS study to unravel the function of EBF1 C-terminus domain

first: Senthilkumar

last: Ramamoorthy

summary: To study the function of the C-terminus domain, DnaseI hypersensitivity assay was performed by transducing Empty, Ebf1wt and Ebf1ΔC expressing vectors into Ebf1-/- progenitor cells.

overall_design: Freshly prepared Ebf1−/− c-Kit+ fetal liver cells were transduced with  pmys-Empty-IRES-GFP, pmys-EBF1wt-IRES-GFP or pmys-EBF1ΔC-IRES-GFP. EBF1wt- or EBF1ΔC-transduced cells were sorted as pro-B cells (GFP+CD19+). vector-transduced cells were sorted as only GFP+.

first: Sören

last: Boller

first: Rudolf

last: Grosschedl


title: An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR

city: Aurora

first: Aaron

last: Johnson

state: CO

department: Biochemistry and Molecular Genetics

institute: University of Colorado School of Medicine

summary: The human long noncoding RNA (lncRNA) HOTAIR acts in trans to recruit the Polycomb Repressive Complex 2 (PRC2) to the HOXD gene cluster and promote gene silencing during development. In breast cancers, overexpression of HOTAIR increases metastatic potential via the repression of many additional genes. It has remained unclear what factors determine HOTAIR-dependent PRC2 activity at specific genomic loci, particularly when high levels of HOTAIR result in aberrant gene silencing. To identify additional proteins that contribute to the specific action of HOTAIR, we performed a quantitative proteomic analysis of the HOTAIR interactome. We found that the most specific interaction was between HOTAIR and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a member of a family of proteins involved in nascent mRNA processing and RNA matchmaking. Our data suggest that A2/B1 are key contributors to HOTAIR-mediated chromatin regulation in breast cancer cells: A2/B1 knockdown reduces HOTAIR-dependent breast cancer cell invasion and decreases PRC2 activity at the majority of HOTAIR-dependent loci. We found that the B1 isoform, which differs from A2 by 12 additional amino acids, binds with highest specificity to HOTAIR. B1 also binds chromatin and associates preferentially with RNA transcripts of HOTAIR gene targets. We furthermore demonstrate a direct RNA-RNA interaction between HOTAIR and a target transcript that is enhanced by B1 binding.  Together, these results suggest a model in which B1 matches HOTAIR with transcripts of target genes on chromatin, leading to repression by PRC2.

overall_design: ChIP-Seq analysis of H3K27me3 in four stable MDA-MB-231 breast cancer cell lines, each with two biological replicates. Cells express the HOTAIR lncRNA or an antisense Luciferase control lncRNA and are +/- lentiviral knockdown of the proteins hnRNP A2/B1

first: A

first: E

last: Meredith

first: M

last: Balas


title: Jmjd2a and Jmjd2c regulate H3K9me3 and H3K36me3 at H3K4me3 positive transcription start sites being essential for ESC self-renewal and embryogenesis

city: Copenhagen N

first: Marianne

middle: Terndrup

last: Pedersen

address: Ole Maaloes Vej 5

department: University of Copenhagen

country: Denmark

institute: BRIC

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

overall_design: Refer to individual Series


title: Jmjd2a and Jmjd2c regulate H3K9me3 and H3K36me3 at H3K4me3 positive transcription start sites being essential for ESC self-renewal and embryogenesis (ChIP-Seq)

summary: We have characterized the role of the Jmjd2/Kdm4 proteins in embryonic stem cell (ESC) biology, histone methylation and gene regulation. The Jmjd2 proteins are H3K9/H3K36 histone demethylases and three Jmjd2 family members are expressed in ESCs: Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1. We find that specifically Jmjd2a and Jmjd2c exert redundant functions, which are essential for ESC self-renewal and early embryonic development. ChIP-seq studies show that Jmjd2a and Jmjd2c both localize to H3K4me3 marked regions, where they have general and widespread roles preventing the accumulation of especially H3K9me3, but also H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity.

overall_design: For the identification of Jmjd2a binding patterns, ChIP experiments were performed using conditional Jmjd2a knockout ESCs (Jmjd2a(f/f);Rosa26::CreERT2). The OHT-treated sample (devoid of Jmjd2a) serves as a negative control. H3K9me3, H3K36me3, H3K4me3 and H3 distributions were mapped for conditional Jmjd2a/c double (Jmjd2a(f/f);Jmjd2c(f/f);Rosa26::CreERT2/CreERT2) and Jmjd2a/b/c triple (Jmjd2a(f/f);Jmjd2b(f/f);Jmjd2c(f/f);Rosa26::CreERT2/CreERT2) knockout ESCs grown in the absence or presence of OHT to induce loss of Jmjd2 expression. ESCs were cultured in 2i serum-free medium.

middle: T

first: Susanne

last: Kooistra


title: Barcode Sequencing Screen Identifies SUB1 as a Novel Regulator of Yeast Mating Pathway Genes

first: Jef

last: Boeke

address: 430 East 29th Street

department: Institute of Systems Genetics

institute: NYU Langone Medical Center

summary: The yeast mating pathway serves as a valuable model of eukaryotic mitogen activated protein kinase (MAPK) pathways, several components of which are actual and potential therapeutic drug targets. Here we present a new screening method we developed that combines two technologies, fluorescence activated cell sorting (FACS) with Barcode analysis by Sequencing (Bar-Seq). Using the new screening method and pFUS1-GFP as a reporter, we identified known mutants in the mating pathway and also characterized a source of false positives that arose as a result of the design of this screen. Additionally from the screen, we identified that a deletion mutant, sub1Δ, that exhibits increased expression of the GFP reporter. Our ChIP-Seq data shows that Sub1 increases binding to the promoters of several pheromone-inducible genes and supports our finding that SUB1 is involved in regulating the expression of genes in mating pathway. RNA-Seq of a sub1Δ mutant shows that the majority of genes have no change in RNA and of the small subset that do, most of these show a decrease in expression. In this study, we find that the sub1Δ mutant has increased basal levels of a small subset of genes including IMD2 (consistent with previous reports) and a gene necessary for efficient mating, FIG1.

overall_design: Bar-Seq of sorted populations of mutants with mutant pFUS1-GFP expression; ChIP-Seq of Sub1-3HA under α-factor and sorbitol conditions; RNA-Seq of sub1Δ mutant under α-factor and sorbitol conditions

first: Anna

last: Sliva

first: Zheng

last: Kuang

first: Pamela

last: Meluh


title: MicroRNA sequencing from ventricles of Sham and TAC mice treated with antimiR-control and antimiR-34

status: Public on May 08 2016

city: Melbourne

first: Jenny

last: Ooi

address: 75 Commercial Road

institute: Baker IDI Heart & Diabetes Institute

summary: Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated in settings of heart disease, and inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy. Under chronic cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identification of transcription factor (TF)-miRNA regulatory networks has added complexity to understanding the therapeutic potential miRNA-based therapies. Here, we sought to determine whether antimiR-34 targets secondary miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identified differentially regulated miRNAs in hearts from mice with cardiac pathology due to transverse aortic constriction (TAC), and these miRNAs were also regulated by antimiR-34. Two clusters of stress-responsive miRNAs were classified as  “pathological” and “cardioprotective”. Using ChIPBase we identified 45 TF binding sites on the promoters of “pathological” and “cardioprotective” miRNAs, and 5 represented direct targets of miR-34, with the capacity to regulate other miRNAs. The expression of two “pathological” miRNAs (let-7e and miR-31) was independently experimentally validated in hearts from antimiR-34 treated TAC mice, and may explain why targeting the entire miR-34 family is more effective than targeting miR-34a alone. AntimiR-34 regulates the expression of other miRNAs and this has significant implications for drug development.

overall_design: MicroRNA-seq profiles in ventricles of mice that develop cardiac hypertrophy induced by transverse aortic constriction (TAC) or sham-operated controls and treated with LNA-antimiR-34 or LNA-control. Four groups of samples were analyzed : Sham LNA-control (n=4), Sham LNA-antimiR-34 (n=3), TAC LNA-control (n=3) and TAC LNA-antimiR-34 (n=4)

middle: Y

first: Bianca

middle: C

last: Bernardo

first: Saloni

last: Singla

first: Ruby

last: Lin

first: Julie

last: McMullen


title: Computationally designed, high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

city: Seattle

first: David

last: Baker

state: WA

address: Molecular Engineering and Sciences, Box 351655

institute: University of Washington

summary: In the related work, proteins were computationally designed and experimentally optimized to bind with high affinity and specificity to each human pro-survival BCL2 homolog. Site-directed saturation mutagenesis libraries were generated based on partially-specific computational designs 2CDP06 (for Bcl-2-targeting Computationally Designed Protein), XCDP07 (Bcl-xL), WCDP03 (Bcl-w), and FCDP01 (Bfl-1), and BCDP01 (Bcl-B) which were then transformed into yeast for surface display, as described in Chao et al (Nat Protoc. 2006). Binding to labeled target homolog was detected via FACS after co-incubation with unlabeled competitors to favor specific interactions. Similarly, an SSM library was generated based on partially-specific Mcl-1-targeting design MCDP02 and was sorted for specific interactions toward each Bcl-2 proteins (data labeled MCDP02_[homolog]). DNA was harvested from naïve libraries and sorted populations and deep sequenced. The frequency of each mutation in a sorted population was compared to its frequency in the naïve population to determine enrichment ratios for all possible single amino acid substitutions (frequency calculations based on counts, which can be found in analyzed data file along with enrichment ratios). The most enriching mutations were then combined in combinatorial libraries, sorted as described above, and variants present after four or five rounds of sorting exhibited excellent specificity. Please see our corresponding publication for additional detail.

overall_design: For all samples except WCDP03, extracted DNA was amplified from two overlapping regions, generating two smaller fragments per target gene, in order to accommodate a 300-cycle MiSeq kit smaller than the length of our genes of interest (approximately 350 base pairs). For each paired-end sequencing experiment, the forward reads each fragment are combined in a single file (*R1*), as are reverse reads (*R2*). For WCDP03, a 600-cycle MiSeq kit was employed, so we instead amplified the entire gene of interest. SSMs based on 2CDP06, XCDP07, WCDP03, FCDP01 and BCDP01 underwent two rounds of FACS and thus three samples exist per construct: naïve library, sort 1 and sort 2. The MCDP02-based library underwent a single round of FACS toward specific binding for each indicated BCL2 homolog, thus there are seven related samples: naïve library plus six sorted populations. In total, there are 22 samples presented here.

first: Stephanie

last: Berger

first: Erik

last: Procko


title: Protracted NP95 binding to hemimethylated DNA disrupts SETDB1-mediated proviral silencing [RRBS-seq]

city: Yokohama

first: Takaho

middle: A.

last: Endo

state: Kanagawa

address: 1-7-22 Suehiro, Tsurumi

department: IMS

institute: RIKEN

summary: Genetic ablation of the maintenance methyltransferase Dnmt1 induces widespread demethylation and transcriptional activation of CpG-rich IAP (intracisternal A particle) proviruses. Here, we report that this phenomenon is not simply a consequence of loss of DNA methylation. By exploiting conditional deletions of Dnmt1 and Np95, each of which is essential for maintenance methylation, we find that while IAPs are indeed de-repressed in Dnmt1-ablated embryos and embryonic stem cells (ESCs), these proviruses remain silenced in Np95-ablated cells, despite similar kinetics of passive demethylation. Paradoxically, transient IAP activation in Dnmt1-ablated ESCs requires the presence of NP95. We subsequently show that in the absence of NP95, the H3K9 methyltransferase SETDB1 maintains IAP silencing; while in the absence of DNMT1, prolonged binding of NP95 to hemimethylated DNA perturbs SETDB1-dependent H3K9me3 deposition. Taken together, these observations reveal that following acute loss of Dnmt1, H3K9 methylation-dependent IAP silencing is disrupted by aberrant NP95 binding to hemimethylated DNA.

overall_design: RNA-seq for Np95, Dnmt1 and Setdb1 wt, single conditional KO (cKO) and double cKO ES cells; RRBS-seq for Dnmt1 and Np95 single and double cKO ESCs; Myc-tagged NP95, DNMT1 ChIP-seq; and wt and Np95wt and cKO H3K9me3 ChIP-seq.

first: Haruhiko

last: Koseki

first: Matthew

last: Lorincz

first: Jafar

last: Sharif


title: A long noncoding RNA lincRNA-EPS acts as a transcriptional brake to restrain inflammation

city: Stanford

first: Howard

last: Chang

state: CA

address: 269 Campus Dr. Stanford, CA

institute: Stanford University

summary: LincRNA-EPS is a long noncoding RNA that is expressed in macrophages and downregulated upon exposure to diverse microbial products. Analysis of macrophages from lincRNA-EPS-deficient mice revealed a specific role for this lincRNA in restraining immune response gene (IRG) expression. Mechanistically, lincRNA-EPS associates with chromatin at regulatory regions of IRGs to repress their transcription. To gain insight into the molecular function of lincRNA-EPS on chromatin, we generated ATAC-Seq libraries from wildtype and lincRNA-EPS-deficient macrophages stimulated with LPS.

overall_design: We examined chromatin structure using ATAC-seq in wild-type and lincRNA-EPS knock-out macrophages stimulated with LPS

first: Maninjay

last: Atianand

first: Ying

last: Shen

first: Ansuman

last: Satpathy

first: Katherine

last: Fitzgerald


title: Protracted NP95 binding to hemimethylated DNA disrupts SETDB1-mediated proviral silencing [ChIP-seq]


title: Multiplex enhancer-reporter assays uncover unsophisticated p53 enhancer logic [ChIP-seq]

status: Public on May 07 2016

city: Leuven

first: Zeynep

last: Kalender Atak

address: Herestraat 49

country: Belgium

institute: KULeuven

summary:
Analysis of p53 binding sites using multiplex enhancer reporter assays, ChIP-seq data and RNA-seq data.

Transcription factors establish and maintain the specific transcriptome of a cell by binding to genomic regulatory regions, thereby regulating the transcription of their target genes. Like many transcription factors, the DNA sequence-specific binding preferences of p53 are known. However, it remains largely unclear what distinguishes functional enhancers from other bound genomic regions that have no regulatory activity. In addition, the genome is scattered with seemingly perfect recognition sequences that remain unoccupied. To disentangle the rules of genome-wide p53 binding, we employed two complementary techniques of multiplex enhancer-reporter assays, one using barcoded reporters and the other using enhancer self-transcription. We compared the activity of more than one thousand candidate p53 enhancers under loss and gain of p53 conditions and identified several hundred high-confidence p53-responsive enhancers. Strikingly, the large majority (99%) of these target enhancers can be characterized and distinguished from negative sequences by the occurrence of a single p53 binding site. By training a machine learning classifier on these data, and integrating the resulting genome-wide predictions with fifteen publicly available human p53 ChIP-seq data sets, we identified a consensus set of 1148 functional p53 binding sites in the human genome. Unexpectedly, this direct p53 cistrome is invariably used between cell types and experimental conditions, while differences between experiments can be largely attributed to indirect non-functional binding. Our data suggest that direct p53 enhancers function in a context-independent manner and do not contain obvious combinatorial complexity of binding sites for multiple transcription factors. They represent a class of unsophisticated cell-autonomous enhancers with a single binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors. This suggests that context-dependent regulation of p53 target genes is not encoded in the p53 enhancer, but at different upstream or downstream layers of the cell's gene regulatory network.

overall_design: Comparing active enhancers between p53 stimulated and non-stimulated MCF7 cells.

first: Annelien

last: Verfaillie

first: Stein

last: Aerts


title: Multiplex enhancer-reporter assays uncover unsophisticated p53 enhancer logic [STARR-seq]

overall_design: STARR-seq enhancer reporter assay to identify and validate p53-responsive enhancers.


title: Multiplex enhancer-reporter assays uncover unsophisticated p53 enhancer logic [CHEQ-seq]

overall_design: CHEQ-seq multiplex enhancer reporter assay to identify and validate p53-responsive enhancers.


title: Multiplex enhancer-reporter assays uncover unsophisticated p53 enhancer logic [RNA-seq]

overall_design: RNA-seq on MCF7 cells with p53 stable knockdown.


title: Multiplex enhancer-reporter assays uncover unsophisticated p53 enhancer logic


title: Transcriptomic responses of mixed cultures of ascomycete fungi to lignocellulose using dual RNA-seq reveal inter-species antagonism and limited beneficial effects on CAZyme expression

city: Nottingham

last: Archer

address: University Park

department: School of Life Sciences

institute: University of Nottingham

summary: Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities.   Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw.   Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement.   This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study.

sample_organism:
Aspergillus niger
Penicillium chrysogenum
Trichoderma reesei

overall_design: 10 conditions were analysed with three biological replicates (30 RNA samples) which were from monocultures of each of the three fungi analysed (with either glucose or wheat straw as carbon source) or mixed cultures with two or three fungal species (with wheat straw as carbon source).

first: Paul

last: Daly

first: Jolanda

last: van Munster

last: Kokolski

first: Fei

last: Sang

first: Martin

last: Blythe

first: Sunir

last: Malla

first: Juliana

middle: V

last: de Castro Oliveira

first: Gustavo

last: Goldman


title: Nuclear Envelope Retention of LINC Complexes Is Promoted by SUN-1 Oligomerization in the Caenorhabditis elegans Germ Line

city: Vienna

first: Cornelia

last: Vesely

address: Schwarzspanierstrasse 17

department: Anatomy

country: Austria

institute: Medical University of Vienna

summary: SUN (Sad1 and UNC-84) and KASH (Klarsicht, ANC-1 and Syne homology) proteins are constituents of the inner and outer nuclear membranes. They interact in the perinuclear space via carboxy-terminal SUN-KASH domains to form the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex thereby bridging the nuclear envelope. LINC complexes sustain numerous biological processes by connecting chromatin with the cytoplasmic force generating machinery. Here we show that the coiled-coil domains of SUN-1 are required for oligomerization and retention of the protein in the nuclear envelope, especially at later stages of female gametogenesis. Consistently, deletion of the coiled coil domain makes SUN-1 sensitive to unilateral force generation across the nuclear membrane. However, absence of this domain does not lead to different expression levels of sun-1 and other known meiotic genes in the mutant compared to wild type. Premature loss of SUN-1 from the nuclear envelope leads to embryonic death due to loss of centrosome-nuclear envelope attachment. However, in contrast to previous notions we can show that the coiled-coil domain is dispensable for functional LINC complex formation, exemplified by successful chromosome sorting and synapsis in meiotic prophase I in their absence.

overall_design: A total number five samples were analyzed including two independent wild-type replicates and three independent mutant replicates by PE 50bp RNASeq.

first: Anahita

last: Daryabeigi


title: RNA-SEQ analysis of fission yeast strains mutant for components of SHREC

status: Public on May 06 2016

city: Memphis

first: David

last: Finkelstein

state: TN

address: 332 N. Lauderdale St.

department: Computational Biology

institute: St Jude Children's Research Hospital

summary:
This project aims to identify molecular effects of loss of individual SHREC components on fission yeast transcriptional program
RNA-seq was utilized to compare transcript levels between wild type, clr1D, clr2D, clr3D, mit1D and chp2D fission yeast

overall_design: Three biological replicate samples from wild type, clr1D, clr2D, clr3D, mit1D and chp2D were used.

first: Brandon

last: Lowe

first: Janet

last: Partridge


title: The Tip60/NuA4 complex is required for proper cell cycle progression and the transition to a post-mitotic state in Drosophila melanogaster

city: Ann Arbor

first: Laura

last: Buttitta

state: MI

address: 5218 Biological Sciences Bldg., 1105 North University Ave.

department: Molecular, Cellular, Developmental Biology

institute: University of Michigan

summary: To get a more global view of the gene expression changes caused by inhibition of Tip60, we performed RNAseq on dissected late L3 wings expressing Tip60WT or Tip60DN. Expression of Tip60WT or Tip60DN was restricted to 48 or 78h prior to RNA extraction. Tip60WT expression caused minimal changes in the transcriptome compared to control wings expressing Gal4/GFP alone, while Tip60DN expression for 48h and 78h led to a largely overlapping program of 860 genes that significantly changed expression >1.4-fold compared to Tip60WT.

overall_design: Transcriptome changes were determined by comparing levels of transcripts in L3 wings expressing a dominant negative Tip60 (Tip60DN) to Tip60WT expression for either 48h or 78h.

first: Kerry

last: Flegel


title: The interaction of PRC2 with RNA or chromatin is mutually antagonistic [RNA-Seq]

city: London

first: Richard

last: Jenner

address: 72 Huntley Street

department: Cancer Biology

institute: UCL Cancer Institute

summary: Polycomb repressive complex 2 (PRC2) maintains developmental regulator genes in a repressed state through methylation of histone H3 at lysine 27 (H3K27me3) and is necessary for cell differentiation. We and others have previously found that the PRC2 subunit Suz12 interacts with RNA in vitro and other studies have shown that Ezh2 and Jarid2 also possess RNA binding function. The interaction of PRC2 with RNA has been suggested to regulate PRC2 targeting or enzymatic activity, but the RNAs directly bound by PRC2 in cells, and the role of each PRC2 RNA binding subunit, remain unclear. We have used different CLIP techniques, which use UV-crosslinking to allow detection of direct Suz12-RNA interactions as they occur in living mouse ES cells. Suz12 binds nascent RNA and has a preference for interaction with the 3’UTR, showing it does have binding specificity in cells. RNAs bound by Suz12 at the 3’UTR encode developmental regulator genes.  Suz12 remains bound to RNA upon deletion of Ezh2 or Jarid2 showing that it binds RNA independently of other PRC2 subunits. We also show that binding of Suz12 to RNA or chromatin is mutually inhibitory. Although Ezh2 and Jarid2 also bind RNA, Ezh2 and Jarid2 deletion causes an increase in Suz12 RNA binding, without changing its specificity, which reflects the loss of Suz12 from chromatin. Similarly, disruption of Suz12-RNA interactions by RNA polymerase II inhibition or RNase treatment increases Suz12 binding to chromatin. These results therefore suggest that Suz12 acts as an RNA sensor, binding to the 3’UTR of nascent RNAs and modulating the interaction of PRC2 with chromatin.

overall_design: Total RNAseq libraires from of Mus musculus Ezh2 fl/fl  Stem Cells after and before Tamoxifen treatment.Up to three replicates per condition

first: Manuel

last: Beltran

first: Christopher

last: Yates

first: Keijo

last: Viiri

first: Lenka

last: Skalska

first: Filipa

last: Reis

first: Cynthia

middle: L

last: Fisher

first: Marcus

last: Dawson

middle: R

last: Sibley

first: Jernej

last: Ule

middle: G


title: Cutoff suppresses RNA polymerase II termination to ensure expression of piRNA precursors

status: Public on May 05 2016

city: Pasadena

first: Maria

last: Ninova

address: 1200 E California Blvd

department: Division of Biology and Biological Engineering

institute: Calfironia Institute of Technology

summary: Small non-coding RNAs called piRNAs serve as the sequence-specific guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. The adaptation of the piRNA pathway to novel transposons is believed to occur when active transposons integrate into piRNA clusters, special genomic regions, which encode piRNA precursors. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) binds chromatin of dual-strand piRNA clusters and is required for transcription of piRNA precursors, though the mechanism by which RDC license transcription remained unknown. Here we show that Cuff prevents premature termination of RNA polymerase II by two distinct mechanisms. First, Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Second, if processing does occur, Cuff protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually novel mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

overall_design: Comparison of RNAseq, GRO-seq, small RNA-seq and Pol II ChIP data between Cutoff knockdown, mutant and control Drosophila ovaries.

first: Yung-Chia

last: Chen

first: Evelyn

last: Stuwe

first: Yicheng

last: Luo

first: Adrien

last: Le Thomas

first: Ekaterina

last: Rozhavskaya

first: Sisi

last: Li

first: Sivani

last: Vempati

first: John

middle: D

last: Laver

first: Dishaw

last: Patel

first: Craig

last: Smibert

first: Howard

last: Lipshitz

first: Katalin

last: Fejes Toth

first: Alexei

last: Aravin


title: The interaction of PRC2 with RNA or chromatin is mutually antagonistic [ChIP-Seq]

first: Richard

last: Jenner

overall_design: Suz12, and Ezh2 ChIP-seq experiments before and after Tamoxifen treatment of Mus musculus Ezh2 fl/fl  Stem Cells.

first: Manuel

last: Beltran

first: Christopher

middle: M

last: Yates

first: Keijo

last: Viiri

first: Lenka

last: Skalska

first: Filipa

last: Reis

first: Cynthia

last: Fisher

last: Dawson


title: Nfib promotes Metastasis through a Widespread Increase in Chromatin Accessibility [RNA-seq]

first: William

last: Greenleaf

address: 279 Campus Drive

summary: We describe Nfib as an important regulator of chromatin accessibility in SCLC.

overall_design:
We performed RNA-seq on three cell lines with either knockdown of Nfib expression or overexpression of Nfib.
Cell lines 16T and KP1 were expressing iether a control vector or a vector expressing shRNA to Nfib, in duplicate
Cell line KP22 were expressing either an empty vector or a vector expressing Nfib cDNA, in duplicate.

first: Sarah

last: Denny

first: Dian

last: Yang

first: Julian

last: Sage

first: Monte

last: Winslow


title: The interaction of PRC2 with RNA or chromatin is mutually antagonistic

summary: Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3’UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.

overall_design: We used individual-nucleotide resolution CLIP (iCLIP) to identify the RNAs directly bound by endogenous PRC2 in mouse embryonic stem cells. We performed iCLIP with an antibody to SUZ12 in 6 biological replicate experiments. In order to relate PRC2 RNA binding activity to other RNA binding proteins and to control for potential biases in the methodology, we performed iCLIP for the splicing regulators FUS and HNRNPC. To control for non-specific crosslink events, we performed iCLIP for SUZ12 in Suz12-/- cells, for GFP in transfected cells and with a non-specific IgG antibody control. Since crosslink frequency is related to RNA abundance, we also performed total RNA sequencing. To determine the role of SUZ12 in PRC2 RNA binding, we also performed iCLIP for SUZ12 in cells lacking EZH2 (Ezh2 d/d) and in cells lack JARID2. RNA-seq was also performed in Ezh2 d/d cells. ChIP-seq was used to measure SUZ12 and EZH2 binding in Ezh2 fl/fl (WT) and Ezh2 d/d cells and to measure changes in SUZ12 and p300 chromatin association upon RNase A treatment.


title: Simultaneous pathway activity inference and global gene expression analysis using RNA-sequencing [SM screen]

city: Boston

first: Raivo

last: Kolde

state: MASSACHUSETTS

address: 185 Cambridge St.

department: Center for Computational and Integrative Biology

institute: Massachusetts General Hospital

summary: Bone marrow derived macrophages treated with small molecules and stimulated with LPS

overall_design: Wild-type BMDMs pretreated with small molecules for 30 minutes prior to stimulation with LPS

first: Daniel

last: O'Connell


title: The interaction of PRC2 with RNA or chromatin is mutually antagonistic [iCLIP-Seq]

overall_design: iCLIP libraries for Suz12 were generated from mESC Ezh2 fl/fl treated with or without tamoxifen for four days to delete Ezh2 in treated cells or  from cells KO for Jarid2 protein.  Several control libraries were generated from mESC Ezh2 fl/fl cells without tamoxifen treatment using General IgG, GFP, hnRNP C or FUS antibody.

first: Marcus

last: Sibley

first: Jernej

last: Ule


title: Transcriptional profiling of JEG3 cells with HLA-G ablation via deletion of Enhancer L

city: Cambridge

last: Mallard

state: MA

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University

summary: HLA-G is a nonclassical HLA molecule expressed specifically in the placenta. While it is known to play a central role in maternal immune tolerance during pregnancy, the mechanism underlying its tissue-specific expression remains poorly understood. To elucidate this mechanism, we used a Massively Parallel Reporter Assay (MPRA) to systematically interrogate the HLA-G locus. This uncovered Enhancer L, a novel cis-regulatory element with enhancer activity 12kb upstream of HLA-G. To verify enhancer function and specificity for HLA-G in culture, we deleted Enhancer L in JEG3 cells and performed RNA sequencing and differential expression analysis. Upon Enhancer L knockout, we observe complete ablation of HLA-G and minimal dysregulation of other genes within 2Mb, suggesting that HLA-G is the only direct cis target of Enhancer L. DNase-seq and Chromatin Conformation Capture (3C) confirm that Enhancer L is a cell type-specific enhancer that loops into the HLA-G promoter. MPRA-based saturation mutagenesis of Enhancer L identifies motifs for transcription factors of the CEBP and GATA families which are essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as a novel mechanism controlling tissue-specific HLA-G expression at the maternal-fetal interface.

overall_design: RNA sequencing of JEG3 cells with CRISPR knockout of Enhancer L

first: Leonardo

last: Ferreira

first: Torsten

middle: B

last: Meissner

first: Tarjei

middle: S

last: Mikkelsen

first: Charles

middle: W

last: O'Donnell

first: Tamara

last: Tilburgs

first: Hannah

last: Gomes

first: Raymond

last: Camahort

middle: I

last: Sherwood

first: David

middle: K

last: Gifford

first: John

last: Rinn

first: Chad

last: Cowan

first: Jack

last: Strominger


title: Transcriptome analysis of H9 hESC derived cerebral organoids

city: La Jolla

first: Tariq

last: Rana

address: 9500 Gilman Dr.

department: Pediatrics

institute: University of California, San Diego

summary: RNA-seq was utilized to characterize the transcriptome of human embryonic stem cells-derived 3D cerebral organoids. Briefly, H9 hESCs were differentiated into cerebral organoids using previously established methods (Lancaster, 2013). Coding and noncoding genes were analyzed in 1 month and 2 month old cerebral organoid samples.

overall_design: Examination of transcriptome of hESC and cerebral organoids 1 month and 2 months old with 2 experimental replicates per group.

first: Jason

last: Dang


title: Simultaneous pathway activity inference and global gene expression analysis using RNA-sequencing [halofuginone]

first: Raivo

last: Kolde

last: O'Connell