title: Novel Observations from Next Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets

status: Public on Jun 08 2015

first: Shaked

last: Afik

address: Stanley Hall

department: Computational Biology Graduate Group

institute: UC Berkeley

summary: Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic a and b cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase b cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify a, b, and d cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the sub-populations by flow cytometry and, using next generation RNA sequencing, we report on the detailed transcriptomes of fetal and adult a and b cells. We observed that human islet composition was not influenced by age, gender, or body mass index and transcripts for inflammatory gene products were noted in fetal b cells. In addition, within highly purified adult glucagon-expressing a cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet a and b cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

overall_design: RNA-sequencing of highly purified human adult and fetal islet cell subset was performed using our newly developed method. Using this data, we can study and compare the detailed transcriptome or alpha and beta cells during development.

first: David

last: Blodgett

first: Anetta

last: Nowosielska

first: Susanne

last: Pechhold

first: Anthony

last: Cura

first: Norman

last: Kennedy

first: Soyoung

last: Kim

first: Alper

last: Kucukural

first: Roger

last: Davis

first: Sally

middle: C

last: Kent

first: Dale

middle: L

last: Greiner

first: Manuel

last: Garber

last: Harlan

first: Philip

last: diIorio


title: RNA-seq for Thermotoga maritima MSB8 glucose evolved cultures

status: Public on Jun 09 2015

city: La Jolla

first: Haythem

last: Latif

state: California

address: 417 Powell-Focht Bioengineering Hall, 9500 Gilman Drive

country: USA

institute: University of California San Diego

type: Expression profiling by high throughput sequencing

summary: RNA-seq was performed on T. maritima wild type, three glucose evolved cultures, and three glycerol adapted cultures. Wild type and glucose evolved strains were grown on glucose minimal media and glycerol evolved cultures were grown on glycerol minimal media. All samples were harvested in exponential phase.

overall_design: Seven samples are included with two biological replicates for each condition.

first: Karsten

last: Zengler


title: Cross-regulation between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa

city: Braunschweig

first: Denitsa

last: Eckweiler

state: Lower Saxony

address: Spielmannstr. 7

department: Institute of Microbiology

country: Germany

institute: Technische Universität Braunschweig

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Two-component systems (TCS) serve as basic stimulus-response coupling mechanisms to allow organisms to sense and respond to a large variety of environmental conditions. In the opportunistic pathogen Pseudomonas aeruginosa, more than 100 genes encode TCS components. To avoid unwanted cross-talk between TCSs, the signaling cascades are usually very specific, with one sensor talking to its cognate response regulator. However, cross-regulation may provide effective means to integrate different environmental stimuli into a harmonized output response and thus to coordinate gene expression in response to complex and challenging habitats. Here, we applied a protein-fragment complementation assay (PCA) and identified a direct interaction of two response regulators of the OmpR/PhoB subfamily, namely PhoB and TctD in P. aeruginosa in vivo. Transcriptional profiling and ChIP-seq analysis uncovered adjacent bipartite binding motifs of the two response regulators in 8 promoter regions, of which 6 showed a common PhoB-TctD binding motif with a fixed spacing between the two motifs. We further demonstrate that phosphate limitation exhibits a PhoB-dependent activating activity on the expression of those genes, whereas the presence of a preferred carbon source inhibits gene expression in a TctD-dependent manner. Thus, the perception and the integration of two important environmental signals are achieved by a titration of the relative amounts of two phosphorylated response regulators that inversely regulate a common subset of genes. In conclusion, our results shed light on the general principles underlying two-component signal transduction pathways and the exploitation of cross-regulation to adapt bacterial behavior to complex environments.

overall_design: Transcriptome profiling of the UCBP-PA14 phoB and tctD mutants complemented by phoB ChIP-seq experiments.

first: Piotr

last: Bielecki

first: Vanessa

last: Jensen

first: Janine

last: Strehmel

first: Tanja

last: Nikolai

last: Häussler


title: piRNA-guided slicing specifies transcripts for Zucchini dependent, phased piRNA biogenesis

status: Public on Jun 07 2015

city: Vienna

first: Dominik

last: Handler

address: Dr. Bohr-Gasse 3

country: Austria

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

type: Non-coding RNA profiling by high throughput sequencing

summary: PIWI-clade Argonaute proteins repress transposable elements in animal gonads. Their sequence specificity is conferred via bound ~23-30nt long piRNAs, which are processed from single stranded precursor RNAs. How transcripts are specified as precursors and processed into stereotypical piRNA populations are central unresolved questions. Here we show that piRNA-guided RNA cleavage in Drosophila results not only in generation of a ping-pong partner piRNA but further triggers efficient 3′ directed and phased primary piRNA biogenesis. Phasing is a feature of primary piRNAs in somatic and germline cells and a consequence of consecutive endo-nucleolytic cleavage events catalyzed by Zucchini. Formation of 3′ and 5′ ends of flanking piRNAs is therefore tightly coupled. Zucchini also participates in 3′ end formation of secondary piRNAs but its function can be bypassed by additional downstream piRNA-guided cleavages and subsequent precursor trimming. Hallmarks of Zucchini-dependent phased piRNA biogenesis are also evident in mouse testes, pointing to an evolutionarily conserved mechanism of piRNA biogenesis.

overall_design: This study aims at understanding how piRNA biogenesis is intiated in the Drosophila germline and understanding the role of the nuclease Zucchini/MitoPLD in piRNA biogenesis in Drosophila/Mouse by analysing small RNA sequencing data of various genotypes and sensor constructs.

first: Fabio

last: Mohn

first: Julius

last: Brennecke


title: Gene expression and epigenetic sexual dimorphism in two jewel wasp species Nasonia vitripennis and Nasonia giraulti [Bisulfite-Seq]

city: Ithaca

first: Andrew

last: Clark

address: 227 Biotech Building

institute: Cornell University

type: Methylation profiling by high throughput sequencing

summary: The extraordinary range in the degree of sexual dimorphism (SD) among animal species is widely perceived to be caused in part by differences in patterns of sexual selection, but sex-specific adaptations and sex chromosome differences also play a role. Studies in insects have discovered a substantial number of sex-biased genes, but little is known about the epigenetic basis of SD. The degree and genome-wide distribution of sex-biased expression become interesting questions in hymenoptera species with haplodiploid sex-determination. To study the genetic and epigenetic architecture of SD and understand the conservation and evolution of sex-biased expression in a haplodiploid system that lacks sex chromosomes, we performed RNA-seq and whole-genome bisulfite sequencing in female and male adult samples of two parasitoid wasp species, Nasonia vitripennis and Nasonia giraulti. More than 75% of the expressed genes displayed significantly sex-biased expression. Both the number and the degree of sex-biased genes are higher than insects like Drosophila melanogaster, which have sex-chromosome mediated sex determination. Females from the two Nasonia species have far more similar expression profiles than does the contrast between the two sexes within either species. Interestingly, the extremely male- and female-biased genes are enriched for totally different functional categories: male-biased genes are highly enriched for key enzymes in sex-pheromone synthesis; female-biased genes are enriched for nuclear-located genes that are responsible for epigenetic regulation of gene expression. Unlike gene expression profiles, DNA methylomes are more similar within species, and no stable differentially methylated genes have been found between the two sexes, suggesting that DNA methylation is not directly responsible for the molecular basis of SD. However, methylation status does influence sex-biased expression: 80% of female-biased genes are methylated, which is more than two-fold higher than the genome average (30%); almost all male-biased and sex-specific genes are non-methylated, which is consistent with the fact that methylated genes have house-keeping functions and a broader expression breadth. Evolutionarily, male-biased genes have greater sequence divergence between the two species, and they are more likely to have a functional paralog in the Nasonia genome. Sex-specific genes have significantly higher non-synonymous substitution rates and dN/dS ratios. In addition, local clusters of sex-biased genes in the genome may have epigenetic properties similar to the sex chromosome. In summary, Nasonia accomplish a striking degree of sex-differential expression through a difference in ploidy along with associated differences in methylations status.

sample_organism:
Nasonia vitripennis
Nasonia giraulti

platform_organism:
Nasonia vitripennis
Nasonia giraulti

overall_design: Whole-genome bisulfite sequencing of 24-hour adult whole body samples of Nasonia vitripennis and Nasonia giraulti using Iilumina sequencing.

middle: G

first: John

middle: H

last: Werren

first: Xu

last: Wang


title: Synergism between PPARα and glucocorticoid receptor signaling promotes self-renewal of BFU-E erythroid progenitors and increases red cell production [ChIP-seq]

city: Cambridge

first: Xiaofei

last: Gao

state: MA

institute: Whitehead Institute for Biomedical Research

summary: ChIP-Seq analyses of GR and PPARa occupancy in mouse E14.5 fetal liver BFU-E cells untreated or treated by DEX with or without GW7647

overall_design: ChIP-Seq on GR and PPAR alpha in purified 10^7 mouse BFU-E cells purified from E14.5 fetal livers with or without treatment of Dexamethasone and/or GW7647

first: Hsiang-Ying

last: Lee

first: M

middle: I

last: Barrasa

first: Hu

last: Li

first: Russell

middle: R

last: Elmes

first: Luanne

last: Peters

first: Harvey

middle: F

last: Lodish


title: Synergism between PPARα and glucocorticoid receptor signaling promotes self-renewal of BFU-E erythroid progenitors and increases red cell production [RNA-seq]

summary: Analyses of gene expression by RNA-Seq in mouse E14.5 fetal liver burst-forming unit erythroid (BFU-E) cells untreated or treated by dexamethasone (DEX) with or without PPARα agonist GW7647.

overall_design: RNA-Seq was performed on enriched populations of mouse BFU-E isolated from E14.5 fetal liver, as well as BFU-E enriched cells treated with Dex ± GW7647.

first: Hsiang

middle: Y


title: Somatic cell fusions reveal extensive heterogeneity in basal-like breast cancer [ChIP-Seq 2]

status: Public on Jun 10 2015

city: Boston

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and it is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells is sufficient to induce luminal-to-basal phenotypic switch implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

overall_design: Examination of histone H3K27me3 modifications in various breast cancer cell lines.

first: Reo

last: Maruyama


title: Synergism between PPARα and glucocorticoid receptor signaling promotes self-renewal of BFU-E erythroid progenitors and increases red cell production

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

overall_design: Refer to individual Series


title: An orthologous epigenetic gene expression signature derived from differentiating embryonic stem cells identifies regulators of cardiogenesis

status: Public on Jun 06 2015

city: Bethesda

first: Brian

last: Busser

state: Md

address: 10 Center Drive

institute: NHLBI/NIH

summary: We report a time course of RNA-seq data from wild-type embryonic stem cells and embryonic stem cells in which the cardiogenic transcription factors ZNF503, ZEB2 and NKX2-5 are depleted with shRNAs differentiating along the cardiac lineage.

overall_design: Biological replicates of RNA-seq data from embryonic stem cells differentiating along the cardiac lineage.

middle: W


title: FOXO1 is required for binding of PR on IRF4, novel transcriptional regulator of endometrial stromal decidualization [ChIP]

status: Public on Jun 05 2015

city: Houston

first: Francesco

middle: J

last: DeMayo

state: Texas

address: 1 Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: We report the whole genome binding profile of FOXO1 and RNA pol II

overall_design: ChIP-seq in decidualizing human endometrial stromal cells

first: Franscesco

first: Yasmin

middle: M

last: Vasquez


title: Gene expression and epigenetic sexual dimorphism in two jewel wasp species Nasonia vitripennis and Nasonia giraulti

first: Andrew


title: Structural evidence for Scc4-dependent localization of cohesin loading

city: Edinburgh

first: Alastair

middle: Robert

last: Kerr

state: Midlothian

address: 2.21 Michael Swann Building, Kings Buildings, Mayfield Road

department: Wellcome Centre for Cell Biology

country: United Kingdom

institute: University of Edinburgh

summary:
Purpose: The goal of this study is to look at differences in genome wide localisation of Scc1 cohesin  between wild type and scc4-m35 in metaphase arrested  cells.
Method: Wild-type (AM1145), wild type constructed in the same way as the mutant strain (carrying a HIS3 marker) (AM15540) and scc4-m35 mutant (AM15537)  cells carrying SCC1-6HA were synchronised with a-factor and arrested in mitosis by treatment with nocodazole/benomyl for 2 hr. Samples were harvested, anti-HA ChIP was performed and  input and IP samples were sequenced for the three strains.
Results: All 16 centromeres were specifically depleted of Scc1 in the scc4-m35 background. Scc1 depletion extended roughly 10 kilobases to either side of the core centromere but not to chromosome arms

overall_design:
ChIP-seq for Scc1-6HA in metaphase-arrested  cells in Wild type and in scc4-m35 mutant (samples VM5-VM10)
Vasso Makrantoni: experiments and Alastair Kerr: ChIP Seq Data analysis
https://github.com/AlastairKerr/Hinshaw2015

first: Vasso

last: Makrantoni


title: Refining brucellosis diagnosis by blood transcriptional profiling.

city: Seattle

first: Scott

last: Presnell

state: WA

address: 1201 Ninth Avenue

institute: Benaroya Research Institute

summary: Diagnosis of brucellosis remains challenging for several reasons, including lack of culture sensitivity, nonspecific symptomatology, and high prevalence of positive serology in endemic areas. The main objectives of this study were to identify blood biomarkers specific to brucellosis compared to other endemic infections and to monitor changes in blood biomarkers during treatment. To obtain a global profile of the disease, we employed RNA sequencing (RNAseq) of whole blood RNA to measure host response against brucellosis infection in patients from Macedonia and Spain. Long-term follow up of patients was used to classify patients as having acute or chronic/reinfection brucellosis and as treatment responders and non-responders. We observed distinct gene expression differences between samples from acute brucellosis and control donors. The magnitude of gene expression changes was associated with antibody titers determined by standard serological tests for brucellosis, including the rose Bengal and standard agglutination tests. The expression signature characteristic of acute brucellosis was also different from that of subjects with leishmaniasis. In depth integration of clinical data and serological findings with our gene expression data will be performed to provide insight into cellular and molecular mechanisms of brucellosis infection.

overall_design: Whole blood from 169 subjects including 105 patients with suspected brucellosis, 17 patients with leishmaniasis, and 47 healthy controls.

first: Prasong

last: Khaenam

first: Mathew

last: Altman

first: Jean-Peirre

last: Gorvel

first: Mile

last: Bosilkovski

first: Ignacio

last: Moriyon

first: Dimitry

last: Popov

first: Kimberly

last: O'Brien

first: Quynh-Anh

last: Nguyen

first: Vivian

last: Gersuk

first: James

last: Eddy

first: Peter

middle: S

last: Linsley


title: Somatic cell fusions reveal extensive heterogeneity in basal-like breast cancer

first: Kornelia

last: Polyak

type: Methylation profiling by genome tiling array


title: Gene expression and epigenetic sexual dimorphism in two jewel wasp species Nasonia vitripennis and Nasonia giraulti [RNA-Seq]

last: Clark

overall_design: Profiling of expression levels in Nasonia vitripennis and Nasonia giraulti adult male and female samples using Illumina RNA-seq

first: John

last: Werren

first: Xu

last: Wang


title: Comparative Characterization of Cardiac Development Specific microRNAs: Fetal Regulators for Future

city: Noida

first: vibha

last: rani

state: UP

address: A-10, sector 62

department: Biotechnology

country: India

institute: Jaypee Institute of Information Technology

summary: MicroRNAs (miRNAs) are small, conserved RNAs known to regulate several biological  processes by influencing gene expression in eukaryotes. The implication of miRNAs as  another player of regulatory layers during heart development and diseases has  recently been explored. The present study was designed to identify the microRNAs  implicated in heart development using next generation sequencing, bioinformatics and  experimental approaches. We sequenced six small RNA libraries prepared from  different developmental stages of the heart using chicken as a model system to  produce millions of short sequence reads. We detected 353 known and 703 novel  miRNAs involved in heart development. Out of total 1056 microRNAs identified, 32.7%  of total dataset of known microRNAs displayed differential expression whereas seven  well studied microRNAs namely let-7, miR-140, miR-181, miR-30, miR-205, miR-103  and miR-22 were found to be conserved throughout the heart development. The 3'UTR  sequences of genes were screened from Gallus gallus genome for potential microRNA  targets. The target mRNAs were appeared to be enriched with genes related to cell  cycle, apoptosis, signalling pathways, extracellular remodelling, metabolic, chromatin  remodelling and transcriptional regulators. The study presents the first comprehensive  overview of microRNA profiling during heart development and prediction of possible  cardiac specific targets and has a big potential in future to develop microRNA based  therapeutics against cardiac pathologies where fetal gene re-expression is witnessed  in adult heart.

overall_design: 6 samples (CHL1, CHL2, CHL3, CHL4, CHL5, CHL6)

first: Yashika

last: Rustagi

first: Vibha

last: Rani


title: Chemical Pull-Down Reveals Comprehensive and Dynamic Pseudouridylation in Mammalian Transcriptome

city: Tianjin

first: Ping

last: Zhu

address: 288, Nanjing Road, Heping District

department: State Key Laboratory of Experimental Hematology

country: China

institute: Institute of Hematology and Blood Disease Hospital

summary: Pseudouridine (Ψ) is the most abundant RNA modification, yet little is known about its content, dynamics and function in mRNA and ncRNA. Here, we perform quantitative MS analysis and develop CAP-seq for transcriptome-wide Ψ profiling. The unexpected high Ψ content (Ψ/U ratio: ~ 0.2% to 0.6%) indicates that pseudouridylation in mammalian mRNA is much more prevalent and comprehensive than previously believed. In concordance, CAP-seq identified 2,084 Ψ sites within 1,929 human transcripts. We prove four previously unknown Ψ sites in rRNA and EEF1A1 mRNA. Genetic and biochemical analysis uncover PUS1 as a major human mRNA Ψ synthase. In response to stimuli, Ψ level and sites are dynamically modulated in stimulus-specific manners. Comparisons between human and mouse pseudouridylation reveal conserved and unique sites across tissue and species. We observe stop codon pseudouridylation and readthrough events simultaneously for HSPB1 mRNA, indicating a role in nonsense suppression. Together, these approaches allow in-depth analysis of transcriptome-wide pseudouridylation events and our comprehensive study provides a resource for functional studies of Ψ-mediated epigenetic regulation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Here we report a transcriptome-wide profiling method that utilizes a chemically synthesized N3-CMC, which pre-enriches the Ψ-containing RNAs and blocks the reverse transcription.Mapping the Ψ sites in human transcriptome was performed using HEK293T and PUS1 dependent Ψ sites were identified by comparing PUS1 knock out cells with wildtype cells. Stress inducible or suppressed Ψ sites were identified by comparing stress treated cells with untreated cells. And mouse brain and liver were used to map Ψ sites in mouse transcriptome.

first: Xiaoyu

last: Li

first: Shiqing

last: Ma

first: Chengqi

last: Yi


title: N6-methyladenosine Modulates Messenger RNA Translation Efficiency

city: Chicago

first: Chuan

last: He

state: Illinois

address: 929 E. 57th. Street

department: Chemistry

institute: The University of Chicago

summary: N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A.

overall_design: PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down

first: X

first: B

last: Zhao

first: I

middle: A

last: Roundtree

first: Z

last: Lu

first: D

last: Han

first: C


title: Epigenetic marks and factors binding to chromatin in mouse embryonic stem cells

first: Jing

last: Huang

state: MD

institute: National Cancer Institute

summary: The goal is to profile several histone modifications and hnRNP L in untreated or Adriamycin-treated mouse embryonic stem cells.

overall_design: Fifty million of R1E mES cells were untreated or treated with 0.5 uM Adriamycin for 8 hours. Fixed cells were subject to ChIP-seq assay.


title: Regulatory RNA Apela Is a Component of a Negative Feedback Loop Regulating p53-Mediated Apoptosis in Embryonic Stem Cells (III)

summary: The goal is to identify RNAs that bind to Hnrnpl (hnRNP L) using RNA immunoprecipitation (RIP) assay.

overall_design: p53+/+ mES cells and p53-/- mES cells were untreated or treated with 0.5 uM Adriamycin for 8 hours. RNA immunoprecipitation was performed using Hnrnpl antibody (Bethyl, Cat#: A311-423A). Total RNA was extracted and subject to RNAseq.


title: Genome-wide maps of SOX9 binding sites in human colorectal cancer cells

first: Toni-Ann

last: Mistretta

state: TX

address: 1 Baylor Plaza, BCM 315

department: Pathology

summary: Aberrant expression of SOX9 in human colorectal cancer cells suggests its roles in the development of colorectal cancer. To gain insight into SOX9-mediated transcriptional regulation in colorectal cancer cells, we attempted to identify its physiological targets on a genome-scale using chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) in HT-29, human colorectal cancer cells.

overall_design: SOX9 CHIP-seq was carried out using HT-29 cells.

first: Yuko

last: Mori-Akiyama

first: Zhongcheng

last: Shi

first: Chi

last: Chiang

first: Toni


title: Regulatory RNA Apela Is a Component of a Negative Feedback Loop Regulating p53-Mediated Apoptosis in Embryonic Stem Cells (II)

summary: The goal is to identify regulatory RNAs that regulate the p53 signaling pathway in embryonic stem (ES) cells.

overall_design: p53+/+ mES cells and p53-/- mES cells were untreated or treated with 0.5 uM Adriamycin for 8 hours. Total RNA was extracted and subject to RNAseq.


title: The LSD1/KDM1A neuro-specific isoform regulates neuronal differentiation through H3K9 demethylation

city: Shanghai

first: Ruitu

last: Lyu

address: 138 Yixueyuan Road Xuhui District

institute: Institutes of Biomedical Sciences Fudan University


title: Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans

city: Zurich

first: Deni

last: Subasic

address: Wintethurerstr. 190

department: Institute of Molecular Life Sciences

country: Switzerland

institute: University of Zurich


title: Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [RNA-Seq]

summary: In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the C. elegans miR-58 miRNA family, comprised primarily of four highly abundant members: miR-58.1, miR-80, miR-81 and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting.

overall_design: RNA was extracted from different miR-58 family mutants (mir-58.1, mir-80; mir-58.1 and mir-80; mir-58.1; mir-81-82) and wild-type Bristol C. elegans strain at late L4 stage and submitted to transcriptome sequencing with Illumina HiSeq2000. The goal was to compare miR-58 target RNA expression and system-wide perturbations across various samples.

first: Anneke

last: Bruemmer


title: Regulatory RNA Apela Is a Component of a Negative Feedback Loop Regulating p53-Mediated Apoptosis in Embryonic Stem Cells


title: The LSD1/KDM1A neuro-specific isoform regulates neuronal differentiation through H3K9 demethylation [ChIP-Seq]

summary: During brain development, histone-modifying enzymes play an important role by orchestrating transcriptional programs that regulate neuronal maturation. Lysine-Specific Demethylase 1 (LSD1, also named as KDM1A) functions as a transcriptional repressor by removing methyl groups at lysine 4 of histone H3 (H3K4). In neurons, alternative splicing can include an additional exon (exon E8a) within LSD1 transcripts, generating a LSD1+8a neuro-specific isoform. We here report that LSD1+8a isoform does not have the intrinsic ability to demethylate H3K4. LSD1+8a functions as a co-activator on its target genes by removing H3K9 repressive histone marks. We identify the supervillin protein (SVIL) as a LSD1+8a interacting partner and demonstrate that SVIL protein regulates neuronal maturation by controlling LSD1+8a mediated H3K9 demethylation. Thus, our results show that alternative splicing provides a genius mechanism by which LSD1 isoforms can acquire a dual specificity (H3K9 vs H3K4) and therefore differentially control specific gene expression patterns during brain development.

overall_design: Examination of 2 different LSD1 isoforms LSD1 and LSD1+8a, LSD1+8a's cofactor SVIL, H3K9me2 in SH-SY5Y cells infected with control or LSD1+8a shRNA during neuronal maturation induced with BNDF at Day0(B0) and Day3(B3).ChIP-seq datas were generated by deep sequencer Hiseq2500.

first: Yang

first: Benoit

last: Laurent

first: Lv

last: Ruitu


title: Transcriptome microRNA profiling of bovine mammary epithelial cells challenged with Escherichia coli or Staphylococcus aureus bacteria reveals pathogen directed microRNA expression profiles

city: Edmonton

first: Guanxiang

last: Liang

state: Alberta

address: 410 Agriculture/Forestry Centre

country: Canada

institute: University of Alberta

summary:
Purpose: The goal of this study is to explore the role of miRNAs in dairy cow response to E. coli and S. aureus, mastitis causing pathogens, is not well understood.
Results: The global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing. A total of 231 known bovine miRNAs were identified with more than 10 counts per million (CPM) in at least one of 13 libraries and 5 miRNAs including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more than 50% of the total reads of known bovine miRNAs. One hundred and fifty novel miRNAs were identified and half of them belong to the bta-miR-2284 family. Seventeen miRNAs were significantly (P<0.05) differentially regulated by the presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6 miRNAs differentially regulated within the first 6 hrs post challenge as compared to one for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (Bta-miR184, miR-24-3p, miR-148, miR-486 and bta-let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and miR-99b) were unique to S. aureus. In addition, our study revealed a temporal differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen differentially expressed miRNAs indicate a significant enrichment in gene ontology functional categories in development/cellular processes, biological regulation as well as cell growth and death. Furthermore, target genes were significantly enriched in several KEGG (Kyoto encyclopedia of genes and genomes) pathways of the immune system, signal transduction, cellular process, nervous system, development and pathways in human diseases, especially cancer.
Conclusion: Using next-generation sequencing, our study identified 150 novel bovine miRNAs and revealed a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development.  E. coli elicited an earlier differential regulation of miRNAs as opposed to a delayed regulation by S. aureus. Furthermore, target gene prediction showed significant enrichments for functions in different biological and cellular processes as well as KEGG pathways in immunity, development and human diseases. Our study provides a further confirmation of the involvement of mammary epithelia cells in contributing to the immune response to infecting pathogens and suggests the potential of miRNAs to serve as biomarkers for diagnosis of mastitis and development of control measures.

overall_design: Bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing, no replicates, using illumina HiScanSQ platform.

first: Weiwu

last: Jin


title: The oxidative demethylase ALKBH3 marks hyperactive gene promoters in human cancer cells

city: Marburg

first: Robert

last: Liefke

state: Hessen

address: Hans-Meerwein-Str. 2

department: Institute of Molecular Biology and Tumor Research (IMT)

institute: Philipps University of Marburg

summary:
Background: The oxidative DNA demethylase ALKBH3 targets single-stranded DNA (ssDNA) in order to perform DNA alkylation damage repair. ALKBH3 becomes up-regulated during tumorigenesis and is necessary for proliferation. However, the underlying molecular mechanism remains to be understood.
Methods: To further elucidate the function of ALKBH3 in cancer, we performed ChIP-seq to investigate the genomic binding pattern of endogenous ALKBH3 in PC3 prostate cancer cells coupled with microarray experiments to examine the expression effects of ALKBH3 depletion.
Results: We demonstrate that ALKBH3 binds to transcription associated locations, such as places of promoter-proximal paused RNA polymerase II and enhancers. Strikingly, ALKBH3 strongly binds to the transcription initiation sites of a small number of highly active gene promoters. These promoters are characterized by high levels of transcriptional regulators, including transcription factors, the Mediator complex, cohesin, histone modifiers and active histone marks. Gene expression analysis showed that ALKBH3 does not directly influence the transcription of its target genes, but its depletion induces an up-regulation of ALKBH3 non-bound inflammatory genes.
Conclusions: The genomic binding pattern of ALKBH3 revealed a putative novel hyperactive promoter type. Further, we propose that ALKBH3 is an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes and thereby plays a role to maintain genomic integrity in ALKBH3-overexpressing cancer cells. These results raise the possibility that ALKBH3 may be a potential target for inhibiting cancer progression.

overall_design: ALKBH3 and IgG ChIP-seq

first: Indra

last: Windhof

first: Jochen

last: Gaedcke

first: Gabriela

last: Salinas-Riester

first: Feizhen

last: Wu

first: Michael

last: Ghadimi

first: Sebastian

last: Dango


title: Optimization of codon translation rates via tRNA modifications maintains proteome integrity

status: Public on Jun 04 2015

city: Münster

first: Danny

middle: D.

last: Nedialkova

address: Von-Esmarch-Str. 54

department: Max Planck Research Group for RNA Biology

institute: Max Planck Institute for Molecular Biomedicine

summary: Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Yeast cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress and accumulate aggregates of endogenous proteins with key cellular functions. Moreover, these cells are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA anticodon modifications in maintaining proteome integrity.

sample_organism:
Saccharomyces cerevisiae
Caenorhabditis elegans

overall_design: Ribosome profiling of wild-type and tRNA modification-deficient yeast and nematodes. Yeast samples were generated in various growth conditions (rich medium versus stress induced by treatment with diamide or rapamycin) and paired mRNA-Seq was performed on a subset of samples. Dataset contains three biological replicates for yeast samples and two biological replicates for nematode samples.

middle: D

last: Leidel


title: High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq using drop-based microfluidics

city: cambridge

first: oren

last: ram

state: MASSACHUSETTS

address: 415 Main Street

department: epigenomics

institute: Broad institute

summary: The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops.  Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.

overall_design:
In this experiment we mixed 2 cell types (mES mEF) and then using single cell novel approach we could be able to find each cell (using its barcode) and assign it to mES of mEF and to produce mES and mEF aggregate bam files (converted to bed for GEO submission).
1152_RNA_RTprimers_Barcodes.txt: A list of all 1152 barcodes sequenced for Read2 fastq files.

first: Assaf

last: Rotem

first: Oren

last: Ram

first: Noam

last: Shoresh


title: Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes

first: Javier

last: Irazoqui

address: 55 Fruit Street, GRJ802

department: Center for the Study of Inflammatory Bowel Disease

institute: Massachusetts General Hospital, Harvard Medical School

summary: We report the profiling of induced mRNA transcripts in two C. elegan replicate populations -- WT (N2) and mutant strain with deficient HLH30. Both strains were fed either OP50 strain of e-coli (normal feed) or S. aureus

overall_design: Examination of infected versus uninfected wildtype and mutant lawns of animals

first: Orane

last: Visvikis

first: Nnamdi

last: Ihuegbu

first: Sid

last: Labed

first: Lyly

last: Luhachack

first: Anna-Maria

last: Alves

first: Amanda

last: Wollenberg

first: Lynda

last: Stuart

first: Gary

last: Stormo

middle: E

last: Irazoqu


title: Next Generation Sequencing Facilitates Quantitative Analysis of miRNAs in hepatic fibrosis and normal tissues.

first: Wang

last: Xiaowei

address: Business park of Tianjin airport economic zone

institute: BGI-Tianjin Techonology Co., Ltd.

summary: Aim: Hepatic fibrosis is a major worldwide medical problem and can develop into liver cirrhosis and hepatocellular carcinoma(HCC). Until now, there are no effective drugs for liver ﬁbrosis because the molecular mechanism of progression of liver fibrosis is not fully understood. MicroRNAs (miRNAs) are an important class of small non-coding functional RNAs that play a key role in many biological processes. The purpose of this study was to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis in rat liver fibrosis model.  Methods: Fibrotic and paired normal liver tissues were collected and assesssed by deep sequencing technology. MiRNA proﬁling results were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and bioinformatics was used to predict miRNA targets.  Results: Nine deregulated miRNAs were induced in porcine serum (PS)-induced hepatic fibrosis versus normal liver. Further analysis revealed several signaling pathways (e.g., gap junction and neuroactive ligand-receptor interaction) may be associated with hepatic fibrogenesis.  Conclusion: Several miRNAs are dysregulated in PS-induced hepatic fibrosis and seem to be closely associated with hepatic fibrogenesis. These results provide an experimental basis for understanding the mechanism of hepatic fibrosis.

overall_design: We sequenced two samples, including case and control. Each sample has two replicates.

first: Xiaowei

last: Wang


title: Control of stomach smooth muscle development and intestinal rotation by transcription factor BARX1 (ChIP-seq)

status: Public on Jun 03 2015

city: New York

first: Chenura

last: Jayewickreme

state: NY

address: 1300 York Avenue

institute: Weill Cornell Medical College

summary: Diverse functions of the homeodomain transcription factor BARX1 include Wnt-dependent, non-cell autonomous specification of the stomach epithelium, tracheo-bronchial septation, and Wnt-independent expansion of the spleen primordium. Tight spatio-temporal regulation of Barx1 levels in the mesentery and stomach mesenchyme suggests additional roles. To determine these functions, we forced constitutive BARX1 expression in the Bapx1 expression domain, which includes the mesentery and intestinal mesenchyme, and also examined Barx1-/- embryos in further detail. Transgenic embryos invariably showed intestinal truncation and malrotation, in part reflecting abnormal left-right patterning. Ectopic BARX1 expression did not affect intestinal epithelium, but intestinal smooth muscle developed with features typical of the stomach wall. BARX1, which is normally restricted to the developing stomach, drives robust smooth muscle expansion in this organ by promoting proliferation of myogenic progenitors at the expense of other sub-epithelial cells. Undifferentiated embryonic stomach and intestinal mesenchyme showed modest differences in mRNA expression and BARX1 was sufficient to induce much of the stomach profile in intestinal cells. However, limited binding at cis-regulatory sites implies that BARX1 may act principally through other transcription factors. Genes expressed ectopically in BARX1+ intestinal mesenchyme and reduced in Barx1-/- stomach mesenchyme include Isl1, Pitx1, Six2 and Pitx2, transcription factors known to control left-right patterning and influence smooth muscle development. The sum of evidence suggests that potent BARX1 functions in intestinal rotation and stomach myogenesis occur through this small group of intermediary transcription factors.

overall_design: We produced lentivirus to express 3xFLAG-BARX1-P2A-EGFP, infected P53DD-immortalized E13.5 stomach mesenchymal cells, and performed ChIP-seq using FLAG antibody

first: Ramesh

last: Shivdasani


title: GLOBAL ANALYSIS OF CIRCULATING AND PLACENTAL MICRORNA SIGNATURES AND PREECLAMPSIA DEVELOPMENT

city: Cuernavaca

first: Veronica

last: Jimenez-Jacinto

state: Morelos

address: Av. Universidad 2001. Col. chamilpa

department: Unidad Universitaria de Secuenciación Masiva y Bioinformática

country: Mexico

institute: Universidad Nacional Autonoma de Mexico

summary: We perform placental microRNA expression profiling to identify potential microRNAs involved in the development and pathogenesis of preeclampsia. microRNA expression profiling for serum was quantified by qPCR and evaluated at 12, 16, and 20 gestational weeks and at the time of preeclampsia diagnosis. Two groups were evaluated using TaqMan Low Density Array plates: a control group with 18 normotensive pregnant women and a preeclampsia group with 16 patients who developed preeclampsia during the follow-up period. The placental microRNA profile was assayed at delivery (7 cases and 7 controls) using the Illumina GAIIx sequencing platform.

first: Margarita

last: Martinez-Fierro

first: Idalia

last: Garza-Veloz

first: Leticia

last: Vega-Alvarado

first: Alejandro

last: Sanchez-Flores

first: Karol

last: Carrillo-Sanchez

first: Cristina

last: Arteaga-Gutierrez

first: Maria

middle: C

last: Aceves-Medina

first: Ivan

last: Delgado-Enciso

first: Olga

last: Barbosa-Cisneros

first: Rosbel

last: Vazquez-Castro

first: Yolanda

last: Ortiz-Castro


title: Widespread Co-translational RNA Decay Reveals Ribosome Dynamics

city: Heidelberg

first: Wu

last: Wei

address: EMBL Heidelberg,Meyerhofstraße 1

department: Genome Biology Unit

institute: EMBL Heidelberg

summary: It is generally assumed that mRNAs undergoing translation are protected from decay. Here, we show that mRNAs are, in fact, co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5′–3′ transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5′ phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress that are dependent on the RNase Rny1. Our approach is simple and straightforward and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns.

sample_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae

overall_design: RNA samples were quantified according to their 5’modification. Samples were quantified using 2 alternative methods. 5PSeq (measures 5’P RNAs) and 5PSeq-fragmented (measures the sequencing bias present in any sample by randomly fragmenting RNA, re-phosphorilating the 5’ends and subjecting the sample to 5PSeq; it is used as a negative control). Different strains of S. cerevisiae and S. pombe samples were analyzed. Cells were grown both in rich media (YPD or YES) and synthetic defined media (SD). Cells were subjected to different treatments such as inhibition of translation elongation with cyclohexamide (CHX), oxidative stress (0.2 mM H2O2) or inhibition of histidine biosyntesys (3-AT; 100mM 3-amino-1,2,4-triazole). S. cerevisiae samples grown in rich media were also analyzed after CHX treatment and sucrose fractionation into monosome and polyribosome fractionation. All samples were analyzed in biological duplicates.

first: Vicent

last: Pelechano

first: Lars

last: Steinmetz


title: SETDB1 Represses Endogenous and Exogenous Retroviruses in B Lymphocytes

city: Saint Louis

first: Patrick

last: Collins

state: MO

address: 1217 Oakleaf dr

department: Pathology and Immunology

institute: Washington University

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

overall_design: Refer to individual Series


title: 1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components

city: Madison

first: Mark

middle: B

last: Meyer

state: WI

address: 1415 Linden Dr.

department: Nutritional Sciences

institute: University of Wisconsin-Madison


title: 1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (ChIP-seq)

summary: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney and skeleton. Interestingly, while several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine the vitamin D receptor (VDR) cistrome in this issue, we conducted a ChIP-seq analysis of binding sites for the VDR across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)2D3. The residual VDR cistrome was comprised of 4617 sites which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)2D3 in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global VDR cistrome regulating a network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.

overall_design: Wildtype mice were fed a standard rodent chow diet (Harlan Teklad, #5008). At 8 weeks of age, 3 mice were treated with 1,25(OH)2D3 (10 ng/g bw) or vehicle control and the proximal half of the small intestine (duodenum and jejunum) was collected 1 h later.

first: Seong

last: Lee

first: Erin

last: Riley

first: Nancy

last: Benkusky

first: Lori

last: Plum

first: Hector

last: DeLuca

first: J

last: Pike


title: Transcriptomes of ΔrelAspoT and ΔdksA mutants of Haemophilus ducreyi in mid-log, transition, and stationary phase

city: Indianapolis

first: Concerta

middle: L

last: Holley

state: IN

address: 635 Barnhill Drive

department: Microbiology and Immunology

institute: Indiana University School of Medicine

summary: To better understand the role of the (p)ppGpp-mediated stringent response in control of H. ducreyi  virulence determinants, here we defined genes potentially regulated by either (p)ppGpp or DksA by using RNA-seq. We were able to show that loss of either (p)ppGpp or DksA resulted in dysregulation of multiple genes including several known virulence determinants. We also show that loss of (p)ppGpp or DksA resulted in differential expression of putative H. ducreyi small RNAs.

overall_design: RNA of Haemophilus ducreyi wildtype, relAspoT and dksA mutants were collected at mid-log, transition, and stationary phases of growth, in quadruplicate using stranded RNA -seq.

first: Stanley

last: Spinola


title: Comparison of human PRDM12 mutants D31Y and E172D with wildtype fibroblasts

city: Vienna

first: Vanja

last: Nagy

address: Dr. Bohr-Gasse 3

institute: IMBA - Institute of Molecular Biotechnology GmbH

summary: Fibroblasts from PRDM12 patients and unaffected wildtype relatives were cultured until near confluency. The transcriptional profile of those cells was determined by mRNA sequencing and uncovered differential expression in several known pain and neurodevelopmental genes.

overall_design: Transcriptome comparison of human PRDM12 mutant and wildtype fibroblasts

first: V

last: Penninger


title: Small RNA profile of pelo1/+ and peloPB60/1 ovaries

city: Beijing

first: huanwei

last: huang

address: 7 Science Park Rd., ZGC life science park

institute: NIBS

summary: The goal of this study is to compare overall small RNA profile in control and pelo mutant ovaries.

overall_design: The small RNA profiles of 3-5 days old pelo1/+ and peloPB60/1 female ovaries were generated by deep sequencing, using Illumina GAⅡ.

first: Fu

last: Yang

first: Rongwen

last: Xi


title: Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [miRNA-Seq]

first: Deni

last: Subasic

overall_design: RNA was extracted from different miR-58 family mutants (mir-58.1, mir-80; mir-58.1 and mir-80; mir-58.1; mir-81-82) and wild-type Bristol C. elegans strain at late L4 stage and submitted to small RNA profiling with Illumina HiSeq2000. The goal was to see whether miR-58 family members can compensate each other's expression.

first: Anneke

last: Bruemmer


title: Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells

city: Seoul

first: Kyu-Tae

last: Kim

address: Irwon-Ro 81

department: Samsung Genome Institute

country: South Korea

institute: Samsung Medical Center

summary: To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors.

overall_design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case.

first: Hye

first: Hae-Ock

first: Kyeung

last: Joo

first: Woong-Yang

last: Park


title: Condensin-Driven Remodeling of X-Chromosome Topology during Dosage Compensation [Hi-C]

city: Berkeley

first: Barbara

middle: J.

last: Meyer

state: CA

address: 16 Barker Hall #3204

department: MCB

institute: HHMI/UCB

summary: The three-dimensional (3D) organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure or how structure influences gene expression. Here we exploit the X-chromosome-wide process of dosage compensation to dissect these mechanisms. The dosage compensation complex (DCC) of C. elegans, a condensin complex, binds to both X chromosomes of hermaphrodites via sequence-specific recruitment sites (rex sites) to reduce chromosome-wide gene expression by half. Using genome-wide chromosome conformation capture and single-cell FISH to compare chromosome structure in wild-type and DCC-defective embryos (DC mutants), we show that the DCC remodels X chromosomes of hermaphrodites into a spatial conformation distinct from autosomes. The dosage-compensated X chromosomes are composed of Topologically Associating Domains (TADs) that have sharper boundaries and more regular spacing than TADs on autosomes. Most TAD boundaries on X coincide with the highest-affinity rex sites, and these boundaries are lost or diminished in DC mutants, thereby restoring the topology of X to a native conformation resembling that of autosomes. Although most rex sites engage in multiple strong DCC-dependent long-range interactions, the strongest interactions occur between rex sites at the DCC-dependent TAD boundaries. We propose the DCC actively shapes the topology of the entire X chromosome by forming new TAD boundaries and reinforcing pre-existing weak TAD boundaries through interactions between its highest affinity sites. Such changes in higher-order X-chromosome structure then influence gene expression over long distances.

overall_design: Our goal was to determine the molecular topology of the dosage compensated X chromosomes of C. elegans.  To do so we performed Hi-C analysis and FISH analysis in wild-type XX embryos and mutant XX embryos in which the dosage compensation complex was defective and could therefore not bind to the X chromosome.   We showed the dosage compensation complex actively shapes the topology of the entire X chromosome and creates a unique, sex-specific Xconformation that differs from the conformation of autosomes. RNA-seq experiments in wild-type and mutant embryos permitted a comparison between changes in chromosome structure and changes in gene expression.

first: Emily

last: Crane

first: Qian

last: Bian

first: Rachel

middle: P

last: McCord

first: Bryan

last: Lajoie

first: Bayly

last: Wheeler

first: Edward

last: Ralston

first: Satoru

last: Uzawa

first: Job

last: Dekker


title: Hitting the caspofungin salvage pathway of human-pathogenic fungi with the novel lasso peptide humidimycin (MDN-0010)

city: Berlin

first: Robert

last: Altwasser

address: Robert-Rössle-Str. 10

department: AG Di Virgilio

institute: Max-Delbrück-centrum

summary: Fungal infections have increased dramatically in the last two decades and fighting infectious diseases require innovative approaches such as the combination of two drugs acting on different targets, or even target a salvage pathway of one of the drugs. The fungal cell wall biosynthesis is inhibited by the clinically used antifungal drug caspofungin. This antifungal activity has been found to be potentiated by humidimycin, a new natural product identified from the screening of a collection of 20,000 microbial extracts and with no major effect when used alone. The analysis of transcriptomes and selected A. fumigatus mutants indicated that humidimycin affects the high osmolarity glycerol response pathway. By combining humidimycin and caspofungin a strong increase of caspofungin efficacy was achieved, demonstrating that targeting different signaling pathways provides an excellent basis to develop novel anti-infective strategies.

overall_design:
Comparison of A.fumigatus samples treated with CAS, Siamycin, and both
Four time points, three replicates. Untreated samples as control

first: Vito

last: Valiante

first: Maria

last: Monteiro

first: Jesús

last: Martín

first: Noureddine

last: El Aouad

first: Ignacio

last: González

first: Olaf

last: Kniemeyer

first: Emilia

last: Mellado

first: Sara

last: Palomoa

first: Nuria

last: de Pedro

last: Pérez-Victoria

first: José

middle: R

last: Tormo

first: Francisca

last: Vicente

first: Fernando

last: Reyes

first: Olga

last: Genilloud

first: Axel

last: Brakhage


title: Condensin-Driven Remodeling of X-Chromosome Topology during Dosage Compensation [ChIP-Seq]

first: Barbara

last: Meyer

first: Emily

last: Crane

first: Qian

last: Bian

first: Rachel

last: McCord

first: Bryan

last: Lajoie

first: Bayly

last: Wheeler

first: Edward

last: Ralston

last: Dekker


title: Condensin-Driven Remodeling of X-Chromosome Topology during Dosage Compensation [RNA-Seq]

first: Job


title: Enhanced Salicylic Acid Biosynthesis Is Involved in Heterosis for Bacterial Defense in Arabidopsis

status: Public on Jun 02 2015

city: Shenzhen

first: Bosheng

last: Li

state: Guangdong

address: SUStech Huiyuan #1 406

department: MCDB

institute: SUSTC (current, may different with previous institution )

summary: We provide evidence that heterosis for bacterial defense exists in hybrids crossed between some Arabidopsis accessions. Comparison of transcriptomes between hybrids exhibiting heterosis for disease resistance and their parents after inoculation with Pst DC3000 revealed that several key genes involved in salicylic acid (SA) biosynthesis were significantly up-regulated in hybrids. Consistently, in response to bacterial infection, more SA [both free SA and SA glycoside (SAG)] accumulated in hybrids compared with both parents. In addition, heterosis for bacterial defense was significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway was blocked. Moreover, we further revealed that increased histone H3 acetylation of the key genes involved in the SA biosynthesis pathway correlated with their up-regulated expression in hybrids.

overall_design: Around 30 inoculated leaves from Arabidopsis accessions Col-0 and Sei-0 as well as their reciprocal hybrids Fcs and Fsc, which showed best-parent heterosis for bacterial defense were pooled in each sample for mRNA Seq using HiSeq 2000 sequencing system (Illumina)

first: Li

last: Yang

first: Xiaoyu

last: Zheng

first: Jigang

first: Guangming

last: He

first: Xinnian

last: Dong

first: Chengcai

last: An

first: Xing

last: Deng


title: Analysis of the transcriptome of  the pulp of a spontaneous late-ripening sweet orange mutant  and its wild type improves our understanding of fruit ripening  in citrus

city: Wuhan

first: Juxun

last: Wu

state: Hubei

address: No.1 of Shizishan street, Hongshan District

institute: Huazhong Agricultral University

summary: Fruit ripening is a complex, genetically programmed process that occurs in conjunction with the differentiation of  chloroplasts into chromoplasts and involves changes to the organoleptic properties of the fruit. In this study, an  integrative analysis of the transcriptome and proteome was performed to identify important regulators and pathways  involved in fruit ripening in a spontaneous late-ripening mutant (‘Fengwan’ orange, Citrus sinensis L. Osbeck) and its  wild type (‘Fengjie 72-1’). At the transcript level, 628 genes showed a 2-fold or more expression difference between  the mutant and wild type as detected by an RNA sequencing approach. At the protein level, 130 proteins differed by  1.5-fold or more in their relative abundance, as indicated by iTRAQ (isobaric tags for relative and absolute quantitation)  analysis. A comparison of the transcriptome and proteome data revealed some aspects of the regulation of  metabolism during orange fruit ripening. First, a large number of differential genes were found to belong to the plant  hormone pathways and cell-wall-related metabolism. Secondly, we noted a correlation between ripening-associated  transcripts and sugar metabolites, which suggests the importance of these metabolic pathways during fruit ripening.  Thirdly, a number of genes showed inconsistency between the transcript and protein level, which is indicative of posttranscriptional  events. These results reveal multiple ripening-associated events during citrus ripening and provide  new insights into the molecular mechanisms underlying citrus ripening regulatory networks

overall_design: Six samples, the WT and MT fruit pulps harvested at 170, 190, and 210 DAF  were subjected to RNA-seq

first: Hualin

last: Yi


title: 1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (RNA-seq)

first: Mark

last: Meyer

summary: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney and skeleton. Interestingly, while several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with either vehicle or 1,25(OH)2D3 and evaluated 6h later. RNA samples from duodena were then subjected to RNA-seq analysis and the data analyzed bioinformatically. 1,25(OH)2D3 altered expression of large collections of genes raised under either dietary condition. 45 genes were found common to both 1,25(OH)2D3-treated groups, and were comprised of genes previously linked to intestinal calcium uptake including S100g, Trpv6, Atp2b1 and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.

overall_design: Male Cyp27b1-/- mice weaned at 3 wk of age were divided into 2 groups of 12 mice each. One group was fed a highly purified vitamin D-deficient diet containing 0.47% Ca, 0.3% P and vitamins A, E, and K (normal diet) while the other was fed the same diet containing 20% lactose, 2% Ca, 1.25% P (rescue diet). At 8 weeks of age, 8 mice per group were administered 1,25(OH)2D3 (2 ng/g body weight (bw)) or vehicle by intraperitoneal injection. Mice were euthanized at 6 h and duodena were collected for RNA-seq analysis. Six samples  per experimental group were prepared.

first: Seong

last: Lee

first: Erin

last: Riley

first: Nancy

last: Benkusky

first: Lori

last: Plum

first: Hector

middle: F

last: DeLuca

first: J

last: Pike


title: Ribosome profiling study of rli1 depeletion strain

city: Bethesda

first: Nicholas

last: Guydosh

address: 8 Center Dr, Bldg 8, 220

department: NIDDK

institute: National Institutes of Health

summary: ABCE1/Rli1 functions as a ribosome recycling factor in vitro, but has not been shown to be crucial for recycling in vivo. We use ribosome profiling and biochemistry to define the role of Rli1 in living yeast. When Rli1 levels were diminished, 80S ribosomes accumulated at stop codons and, surprisingly, in the adjoining 3'UTRs of most genes. While ribosomes did not show preference for any reading frame in the 3'UTR, their enrichment at stop codons and His codons after histidine starvation is consistent with aberrant 3'UTR translation. Predicted 3'UTR translation products were detected by Western analysis and mass spectrometry, and their small sizes indicate a reinitiation mechanism. Eliminating ribosome-rescue factor Dom34 dramatically increases 3'UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and for overall gene expression as it controls ribosome homeostasis and 3'UTR translation.

overall_design: 10 biological samples are included in the study (2 mRNA-Seq samples and 8 ribosome footprinting samples). These include wild-type, rli1-depletion, rli1-depletion/dom34Δ, and RLI1 high-copy / dom34Δ strains. Samples are also included where cells were treated with 3-AT to starve for histidine.

first: David

last: Young

first: Fan

last: Zhang

first: Alan

middle: G

last: Hinnebusch

first: Rachel

last: Green


title: The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells

city: Hangzhou

first: Hongshan

last: Guo

state: Zhejiang Province

address: No.1369, Wenyixi Road

department: School of Medicine, Zhejiang University

institute: Liangzhu Laboratory

summary: Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10-11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation; with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.

overall_design: In total, we analyzed the transcriptomes of 233 individual male and female human PGCs from 15 embryos at between 4 and 19 weeks of gestation as well as 86 neighboring somatic cells from 13 of these embryos using a single-cell RNA-Seq method that we developed. Furthermore, we analyzed the DNA methylomes of both male and female human gonadal PGCs as well as the neighboring somatic cells of eleven of these embryos at between 7 and 19 weeks of gestation using whole-genome bisulfite sequencing (WGBS). In total, we generated 1.3 billion 100 bp paired-end reads for transcriptome analyses and 4.1 billion 100 bp or 150 bp paired-end reads for DNA methylome analyses.

first: Lin

last: Li

first: Fuchou

last: Tang


title: Condensin-Driven Remodeling of X-Chromosome Topology during Dosage Compensation

first: Barbara


title: PGC reversion to pluripotency involves erasure of DNA methylation from imprinting control centers followed by locus-specific re-methylation

status: Public on Jun 01 2015

city: Los Angeles

first: Juehua

last: Yu

address: 615 Charles E. Young South BSRB 457

institute: UCLA

summary: Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo.  However when removed from their embryonic niche PGCs undergo reversion to generate pluripotent embryonic germ cells (EGCs) in vitro.  One of the major differences between EGCs and embryonic stem cells (ESCs) involves variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood.  In the current study we show that reverting PGCs to EGCs involves ICC methylation erasure, which remain stably hypomethylated at Snrpn, Igf2r and Kcnqot1.  In contrast, the H19/Igf2 ICC undergoes almost complete de novo remethylation. Using the same approach for PGCs differentiated in vitro from ESCs we show that the Snrpn ICC is erased however the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC is abnormally hypermethylated in ESCs, ICC methylation is not erased with differentiation into PGCs.  This highlights the importance of not only launching germline differentiation with correctly methylated ESC lines but also the need to better stabilize the hypomethylated state in the in vitro derivatives following ICC erasure.

overall_design: RNA seq of E9.5 PGCs, iPGCs, PGCLCs and EGCs using small cell numbers from start. N=2 biological replicates in 2 technical sequencing replicates.

first: Ziwei

first: Amander

middle: T

last: Clark


title: Transcriptional perturbations by long noncoding RNAs with distinct spatio-temporal expression in the mammalian brain

city: Cambridge

first: Abbie

last: Groff

state: MA

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology;

institute: Harvard University

summary:
Long noncoding RNAs (lncRNAs) have been implicated in numerous cellular processes including brain development. Yet the in vivo expression dynamics and molecular pathways regulated by these molecules are less well understood. Here, we leveraged a cohort of 13 lncRNA null-mutant mouse models to investigate the spatio-temporal expression of lncRNAs in the developing and adult brain. We observed a   wide range of different spatio-temporal expression profiles in the brain. Several lncRNAs are differentially expressed both in time and space, and others present highly restricted expression in only selected brain regions. We further explore the consequent transcriptome alterations after loss of these lncRNA loci, and demonstrate altered regulation of a large variety of cellular pathways and processes. We further found that 6/13 lncRNA null-mutant strains significantly affect the expression of several neighboring protein-coding genes, in a cis-like manner. This resource provides insight into the expression patterns and potential effect of lncRNA loci in the developing and adult mammalian brain, and allows future examination of the specific functional relevance of these genes in neural development, brain function, and disease.
We have sequenced wildtype and mutant whole brains from a cohort of 13 lncRNA knockout mouse strains at two developmetal timepoints (E14.5 and adult).

overall_design:
Comparison between wildtype and mutant whole brains transcriptomes in 13 lncRNA mutant strains at two different timepoints.

Please note that for each knockout strain there are KO_E14.5 and KO_Adult samples, however for WT, each KO strain was compared to a cohort of 14 WTs (N3 background) and 3 WTs (N2.5 background) at either Adult or E14.5 timepoint. So in total there are 14 WT_Adult and 14 WT_E14.5 and in each differential analysis the 2 or 3 KOs (in N3 background) were compared to this entire cohort at the respective timepoint;  a cohort of 3 WT_adult (N2.5) or 3 WT_E14.5 samples compared to other N2.5 KO samples at the respective timepoint. Thus, each processed data file was generated by comparing each KO strain to a cohort of WTs (at either Adult or E14.5 timepoint; ko_vs_WT_Adult or ko_vs_WT_embryonic).

The mouse strain (background) used in these experiments a cross between 129 and C57BL/6 in the third generation (N3) of breeding in the C57BL/6 line, with the exception of the KANTR mice, which are N2.5.

first: L

last: Goff

first: A

first: M

last: Sauvageau

last: Rinn


title: Methylation profiling of hydatidiform moles

first: Desheng

last: Gong

address: No.7 PengFei road

institute: Agricultural Genomes Institute at Shenzhen


title: Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects.

city: Shanghai

first: Fengjuan

state: Shanghai

address: 320 Yueyang Road

department: Institute of Biochemistry and Cell Biology

institute: Shanghai Institutes for Biological Sciences, CAS

summary: RNA interference (RNAi) is an evolutionarily conserved phenomenon of post-transcriptional gene silencing mediated by small interfering RNAs (siRNAs) generated from short hairpin RNAs (shRNAs) by Dicer cleavage. Here, we report that siRNA precursors can be divided into two categories with different processing mechanisms and silencing activities that are dependent on stem loop length. We designed an alternative siRNA precursor for triggering RNAi named single-stranded Argonaute2 (Ago2)-processed interfering RNA (saiRNA). saiRNA is composed of a 16-18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleavage ribozyme derived from hepatitis delta virus (HDV) to the 3’ end of the saiRNA dramatically improved its silencing activity. Unlike classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during its processing not only eliminated the passenger strand but also avoided the association of mature siRNA with non-nucleolytic Ago proteins, thereby further reducing the risk of off-target effects. Additionally, saiRNA exhibited less competition with the biogenesis of endogenous miRNAs. Therefore, HDV ribozyme-enhanced saiRNA provides a reliable tool for RNAi applications.

overall_design: A total of 400 ng plasmids of control, shRNA, shRNA-LC, saiRNA-RZ were transiently transfected into HEK293 cells in a 6-well plate for 48 hr. For RNA-seq, total cellular RNA was extracted to construct a cDNA library using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) according to the manufacturer’s protocol. The cDNA libraries were sequenced using Illumina Hi-seq 2000 with 2 × 100 running circles. For small-RNA seq, total cellular RNA was extracted to construct a small RNA library according to the Illumina protocol,  and cDNA libraries was sequencing by Hi-seq 2000 with 50 running circles.

first: Renfu

last: Shang

first: Beiying

last: Xu

first: Hairui

last: Xi

first: Xue

first: Weihua

last: Wang

first: Ligang

last: Wu


title: Genome-wide DNA methylation analysis of diploid hydatidiform moles with androgenetic or biparental genomic origin

summary: Only very few studies have investigated methylation patterns of different types of hydatidiform moles (HMs). Methylation patterns of androgenetic HMs (AnHMs) are abnormal due to the fact that the nuclear genome in AnHMs is inherited from the father, only. Diploid biparental HMs (BiHM) have been suggested to display the same methylation patterns of imprinted genes as AnHMs, and the methylation patterns are suspected to be a consequence of a failure to establish maternal methylation at multiple genome-wide loci. We have investigated the methylation patterns of AnHMs, BiHM-like placentas with a chr. 11p15.5 deletion and a BiHM from a woman with NLRP7 mutations and compared these to methylation patterns of normal placentas. Using the Next Generation Sequencing (NGS) technique Reduced Representation Bisulfite Sequencing (RRBS) we instigated the genome-wide CpG methylation of 32 samples, including nine normal placentas, 20 androgenetic diploid HMs (AnHMs), and three diploid biparental HMs/HM-like placentas. This dataset contains RRBS data from 12 samples, including the nine normal placentas and the three diploid biparental HMs/HM-like placentas.The RRBS data from 20 androgenetic diploid HMs (AnHMs) was deposited in GSE65881:http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65881

overall_design:
A total of 32 human placentas were analysed. Nine normal placentas were used as negative controls in comparison with 20 androgenetic diploid HMs (AnHMs), and three diploid biparental HMs/HM-like placentas. The biparental HMs/HM-like placentas were of two different types: One BiHM originated from a woman that were homozygous for a splice-site mutation in NLRP7 and that had familial recurrent BiHM (sample131,BiHM/NLRP7mut). Two biparental HM-like placentas were both heterozygous for a terminal deletion on chr. 11 with breakpoint in 11p15.5 (sample101 and sample635, BiHM/del11p15.5).
GSE67412 represents the RRBS data from 9 normal placentas and three diploid biparental HMs/HM-like placentas.

first: Lotte

last: Andreasen

first: Xiaoyu

last: Pan


title: The microRNA-212/132 cluster regulates hematopoietic stem cell maintenance and survival with age by buffering FOXO3 expression

city: Stanford

first: Georgi

middle: Kolev

last: Marinov

state: California

address: 279 Campus Drive West, Beckman Center, B-257A/259

department: Genetics

institute: STANFORD UNIVERSITY

summary: MicroRNAs are critical post-transcriptional regulators of hematopoietic cell-fate decisions, though little remains known about their role in aging hematopoietic stem cells (HSCs). The microRNA-212/132 cluster (miR-212/132) is enriched in HSCs and is up-regulated during hematopoietic aging. Both over-expression and deletion of microRNAs in this cluster leads to inappropriate hematopoiesis with age. Enforced expression of miR-132 in the bone marrow compartment of mice led to rapid HSC cycling followed by their depletion. A genetic deletion of the miR-212/132 cluster in mice resulted in HSCs that had altered cycling, function, and survival in response to growth factor starvation. We found that miR-212/132 exerts its effect on aging HSCs by targeting the transcription factor FOXO3, a known aging associated gene. Our data demonstrates that miR-212/132 plays a role in maintaining balanced hematopoietic output by buffering FOXO3 expression. We have thus identified a novel target that may play a role in age-related hematopoietic defects.

overall_design: RNA-seq (SMART-Seq2 protocol) for LT-HSCs (LSK CD150+ CD48-), ST-HSCs (LSK CD150- CD48-) and MPPs (LSK CD150- CD48+) from WT and miR-212/132-/- mice

first: Arnav

last: Mehta

middle: K

first: David

last: Baltimore


title: The RNA polymerase II CTD phosphatase SSUP-72 regulates alternative polyadenylation in C. elegans neurons.

city: Ann Arbor

first: Ryan

last: Mills

state: MI

address: 100 Washtenaw Ave

department: Computational Medicine and Bioinformatics

institute: University of Michigan

summary: Alternative polyadenylation (APA) plays important roles in generating transcriptomediversity. In the nervous system widespread APA are observed in development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. Through systematic genetic studies and genome-wide survey of transcriptional landscape, here, we report a mechanism controlling APA of two neuronal genes, UNC-44/Ankyrin and DLK-1/MAPKK, during development of C. elegans nervous system. We show that SSUP-72, a Ser5 phosphatase for RNA polymerase II (Pol II) Carboxyl-Terminal Domain (CTD), differentially affects the usage of poly(A) sites (PASs) in the coding regions of unc-44 and dlk-1. A novel nuclear protein SYDN-1 interacts with both SSUP-72 and RNA pol II, and inhibits the function of SSUP-72 and nuclear polyadenylation factors. This regulatory pathway dampens the usage of a strong internal PAS of unc-44 for the production of neuron-specific ankyrin, and promotes the usage of a weak PAS of dlk-1 to control its activity. Deregulation of unc-44 and dlk-1 mRNA isoforms impairs neuronal development. Our data illustrate the high degree selectivity of APA regulation in complex nervous systems.

overall_design: Two (2) wildtype N2 libraries and two (2) sydn-1 mutant libraries.

first: Fei

last: Chen

first: Yu

last: Zhou

first: Yingchuan

last: Qi

first: Hairi

last: Li

first: Ting

last: Han

first: Sang

middle: Y

last: Chun

first: John

last: Kim

first: Xiang-Dong

last: Fu

first: Yishi

last: Jin


title: Redundant AGO-hook platforms and AGO4-DNA interaction in polV-directed RdDM

city: Seattle

first: Christopher

middle: Joel

last: Hale

state: WA

address: 1959 NE Pacific St., HSC H-458

department: Pathology

institute: University of Washington

summary: Small RNA-dependent pairing of AGO/PIWI-containing effector complexes to chromatin-bound nascent transcripts has been proposed as a universal mechanism for guiding RNA-mediated TGS in eukaryotes. Likewise, Pol V-dependent transcripts have been implicated in the targeting of AGO4 to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we show that the AGO hook platforms of PolV and SPT5L, another component of the transcriptional complex, are functionally redundant yet essential for RdDM at a genome-wide level. Synthesis of Pol V transcripts is uncoupled from AGO4 recruitment in AGO hook-minus plants, challenging the prevailing RNA-based mechanism of AGO4 targeting to chromatin in RdDM. Transcription is essential to lock the PolV transcription complex into a stable and productive DNA-bound state potentiating interactions of AGO4 with DNA. Consistent with this idea, laser UV-assisted crosslinking shows specific AGO4-DNA interaction at RdDM loci, suggesting a revised model for Pol V-mediated DNA methylation in plants, which explains the exquisite specificity of methylation.

overall_design: Three sequencing replicates (denoted 131213, 140123, and 140506) were carried out for wild-type, mutant, and transgenic lines. The data from each replicate was merged for each genotype to generate processed data files and the main figures from the study.

middle: J


title: Drosophila ovarian stage 8 and 10 follicle gene expression

city: Baltimore

first: Allan

last: Spradling

state: MD

address: 3520 San Martin Dr.

department: Embryology

institute: Carnegie Institution/HHMI

summary: We identified genes whose expression changes between stage 8 and stage 10. We also identified genes whose expression was altered in stage 10 from temperature senstive ecdysone receptor mutant flies raised at the restrictive temperature. The experiments showed a significant number of genes that are upregulated in stage 10 depend EcR-mediated signaling.

overall_design: 3 samples x 3 repeats

first: Matthew

last: Sieber


title: Laser capture microdissection/RNA-Seq analyses of Hoxa9,10,11/Hoxd9,10,11 mutant limb development

city: Cincinnati

first: Steven

last: Potter

state: OH

address: 3333 Burnet Ave.

department: Developmental Biology

institute: Children's hospital Medical Center

summary: We used a novel recombineering method to introduce frameshift mutations into the first exons of the flanking Hoxa9,10,11 and Hoxd9,10,11 genes. By interbreeding it was possibleto make mice homozygous mutant for all six genes. These mice showed an extreme shortening of the zeugopod, with only tiny cartilaginous remnants of the ulna and radius made.We used laser capture microdissection to isolate the cells of the developing mutant zeugopods. Mutant and wild type samples were compared by RNA-seq. The results define the many downstream functional pathways perturbed by the Hox mutations, including the upregulation of many genes known to inhibit chondrocyte and osteoblast differentiation, BMP signalin, and growth arrest and apoptosis. In addition we observed the downregulation of genes required for WNT signaling and chonrdocyte maturation.

overall_design: RNA-seq comparison of Hoxa9,10,11/Hoxd9,10,11 mutant and wild type developing limbs in mouse.Also includes different compartments of wild type developing limb.

first: Anna

middle: M

last: Raines

first: Bliss

last: Magella

first: Michael

last: Adam

first: S

middle: S


title: Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis

city: Wuhan City

first: JIE

last: YE

state: Hubei Province

address: No. 1, Shizishan Road

institute: Huazhong Agricultural University

summary:
Purpose: Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. The goals of this study are to investigate dynamic changes of tomato genes during fruit development at transcription level and provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.
Methods: The transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. Raw sequences were filtered and the resulting sets of clean reads were used for the following analysis by tophat and edgeR software.  qRT–PCR validation was performed using  SYBR Green assays
Results: The average number of reads produced for each sample was 9.5 million, with the number of clean reads per sample ranging from 3.3 to 10.9 million. The number of reads that were mapped to the S. lycopersicum genome ranged from 1,786,380 to 6,780,667. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis and 15 of these were validated with qRT–PCR. Correlation analysis revealed a high degree of consistency between transcript abundance determined by qRT-PCR or RNA-seq.
Conclusions: Using RNA-seq analysis, the transcript abundance of a total of 26,397 genes was revealed. A total of 823 transcription factors were identified and their expression levels were compared to those of genes encoding enzymes involved in flavonoid, ascorbic acid and carotenoid biosynthesis. This revealed 20, 34 and 37 transcription factors putatively involved in the biosynthesis of flavonoids, ascorbic acid and carotenoids, respectively. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.

overall_design: Investigate the transcriptome changes during seven fruit developmental stages of two tomato cultivars by Illumina sequencing platform.

first: Yuyang

last: Zhang

first: Jie

last: Ye


title: Mycobacterium tuberculosis transcriptome on various phases of dormancy in potassium-deficient model

first: Dmitriy

last: Ignatov

address: Charitéplatz 1

institute: Max Planck Unit for the Science of Pathogens

summary: Dormant Mycobacterium tuberculosis bacilli play important role in latent tuberculosis infection. Previously we have demonstrated that cultivation of M. tuberculosis in potassium-deficient media resulted in the generation of dormant ‘non-culturable’ cells. Addition of a moderate concentration of rifampicin enabled to kill a minor subpopulation of actively replicating bacilli and obtain a homogeneous ‘zero-CFU’ population of dormant cells characterized by total inability to produce colonies on solid media and by high potential to reactivate after reintroducing of potassium. An RNA-seq based transcriptome analysis of this dormant ‘zero-CFU’ population revealed a 30-50 fold decrease of the total level of mRNA in the cells, indicating global shift-down of gene expression level. The residual scarce protein-coding transcriptome of the dormant cells showed decreased abundance of mRNAs encoding ribosomal proteins and enzymes of TCA cycle and respiratory chain, and increased abundance of mRNAs encoding PE-PGRS proteins. Interestingly, the transcriptome of dormant cells showed little changes during several days of persistence. This stability of ‘dormant’ transcripts may reflect their readiness for translation upon resuscitation process. Transition of M. tuberculosis cells to dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point, located outside the ribosome catalytic center. Another feature of the ‘dormant’ transcriptome was an increased abundance of non-coding transcripts. Enrichment of "dormant transcriptome" by small non-coding RNAs playing regulatory function in the cell probably indicates their role in transition to and maintenance of dormant ‘non-culturable’ state.

overall_design: Twelve samples, representing Mycobacterium tuberculosis H37Rv cells at various time points in the model of dormancy: "Log", "DE", "DM" and "DL". Each of the the time points is represented by three independently sequenced biological replicates.

first: Elena

last: Salina

first: Mikhail

last: Fursov

first: Timofey

last: Skvortsov

first: Tatyana

last: Azhikina

first: Arseny

last: Kaprelyants


title: Analysis of Nestin-GFP+ pericytes from adipose tissue: PDGFRa wild type versus PDGFRa+/D842V (constitutively active mutant)

city: Richmond

last: Dozmorov

state: VA

address: 830 E Main St

department: Biostatistics

institute: Virginia Commonwealth University

summary: Analysis of Nestin-GFP+ pericytes flow sorted from 3-day-old mouse cutaneous adipose tissue, comparing controls with wild type PDGFRa, and mutants with increased PDGFRa signaling driven by a Cre/lox-inducible D842V knockin mutation in the PDGFRa kinase domain. The control cells have adipogenic properties in vitro or when transplanted subcutaneously into recipient mice. The D842V mutant cells show altered behavior in the same assays, with poor adipogenic differentiation but a propensity to transition into profibrotic cells that secrete collagen

overall_design: 3 Nes-GFP+ cells samples; 3 Nes-GFP;Nes-Cre;PDGFRα+/[S]D842V samples

first: Lorin

middle: E

last: Olson

first: Tomoaki

last: Iwayama


title: Genetic, Molecular and Physiological Basis of Variation in Gut Immunocompetence

city: Lausanne

first: Maroun

last: Bou Sleiman

state: Vaud

address: Rue Cantonale

department: IBI

institute: EPFL

summary: Gut immunocompetence involves immune, stress, and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, we perform enteric infection of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila and observe extensive variation in survival. Using genome-wide association analysis, we identify several novel immune modulators. Transcriptional profiling further shows that the intestinal molecular states of resistant and susceptible lines differ, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study provides novel insights into the determinants underlying population-level variability in gut immunocompetence, revealing how relatively minor, but systematic genetic and transcriptional variation can mediate overt physiological differences that determine enteric infection susceptibility.

overall_design: RNA-sequencing of whole guts of female adults from different lines of the Drosophila Genetic Reference Panel (DGRP) was performed under two conditions: fed on sucrose or infected with Pseudomonas entomophila.

first: Dani

last: Osman

first: Andreas

last: Massouras

first: Ary

middle: A

last: Hoffmann

first: Bruno

last: Lemaitre

first: Bart

last: Deplancke


title: Altered DNA Methylation of MAP3K6 and HPCAL1 as Potential Predictors for Persistent Trophoblastic Disease

first: Desheng

last: Gong

summary: Patients with molar pregnancy are at risk of developing persistent trophoblastic disease (PTD). Previous study indicated that PTD risk was not related to evacuation time of hydatidiform mole (HM), leading to our hypothesis that HM might be pre-programmed before evacuation to determine the PTD risk. Considering the similarity between PTD and tumorigenesis, we investigated the methylation alterations in PTD conceptus using reduced representation bisulfite sequencing (RRBS), in order to identify potential predictors for PTD.

overall_design: 10 persistent trophoblastic disease (PTD) samples were chosen on the basis of the karyotype (46, XX), parental origin (P1P1) of the mole genome and the gestational age at evacuation between 9 and 12 weeks. 10 non-PTD samples were chosen upon the same criteria. Another 22 samples (10 PTD versus 12 non-PTD) were collected for validation experiment. Data on clinical features and chemotherapy were collected from gynecologic and oncologic hospital records. All patients had at least 6 months of follow-up from evacuation date, and the median follow-up time was 10.4 years. The regional Committee on Biomedical Research Ethics approved the study and all patients gave informed consent.

first: Xiaoyu

last: Pan


title: Multiplexing of ChIP-seq samples for a model experimental condition has minimal impact on peak detection

city: new york

first: Thadeous

middle: James

last: Kacmarczyk

state: ny

address: 1300 York Ave

department: Medicine/Hematology-Oncology

institute: Weill Cornell Medicine

summary:
ChIP-seq experiments are standard experimental procedure for interrogating epigenetic states and protein-DNA interactions. Sequencing experiments are often designed according to the trade-off between the need to obtain maximum sequencing coverage limited funds. Multiplexing samples is a common approach to minimize cost and maximize information yield. We therefore performed an extensive ChiP-seq multiplexing study to gain a better understanding of the effect of multiplexing on the resulting peak detection and genomic annotation and to provide solid guidelines for multiplexing ChIP-seq studies. For a well characterized antibody, our results indicate that multiplexing to ~20M reads (roughly 8 samples per sequencing lane) is sufficient to capture most of the biological signal.
Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed an analysis of peak detection to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million reads each and still detect over 90% of all peaks found when using a full lane for sample. Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

overall_design: We devised a multiplexing titration scheme starting from one full lane of ChIP and one lane of input samples down to 7 IP samples and one input in a single lane (Figure 1). The remainder of the flow cell contained two lanes of two ChIPs and two inputs, one lane of 5 ChIPs and one input, and one lane of 7 ChIPs and one input.

first: Caitlin

last: Bourque

first: Xihui

last: Zhang

first: Yanwen

last: Jiang

first: Yariv

last: Houvras

first: Alicia

last: Alonso

first: Doron

last: Betel