title: A long non-coding RNA tunes PRC1 function during human and zebrafish development

first: Robert

middle: E

last: Kingston

state: Massachusetts

address: 185 Cambridge Street

department: Molbio

institute: MGH/Harvard

summary:
Long non-coding RNAs (lncRNAs) are emerging as functional regulators of gene expression in many species1,2. However, aligning lncRNA-dependent phenotypes with their underlying molecular mechanisms remains challenging. The Polycomb Repressive Complex 1 (PRC1) family of the Polycomb Group (PcG) complexes interacts with lncRNAs and is essential for gene silencing during development. Despite the functional importance of PRC1, few specific lncRNAs that guide PRC1 activity are known3-5. We screened for human RNAs which co-precipitate with the PRC1-component BMI1, from a chromatin fraction. Knockdown of several of the lncRNA candidates perturbed PcG-regulated gene expression in vivo. In particular, we found that the PRC1-associated lncRNA CAT7 tunes PRC1 function during zebrafish and human development. During neuronal differentiation of human embryonic stem cells, CAT7 impacts expression and PcG-binding of the MNX1 locus, which encodes a master regulator of motor neuron development. During zebrafish development, human CAT7 functionally rescues defects caused by interference of the non-syntenic analog, zebrafish cat7 (zcat7.) Further, zcat7 genetically interacts with bmi1a/b during zebrafish embryogenesis. We propose that PRC1 acts in concert with specific lncRNAs, and that CAT7/zcat7 represent lncRNAs that convergently evolved to tune PRC1 repression at individual loci.

We immunoprecipitated PRC1-interacting RNAs from a chromatin fraction using a technology called GRIP.  We identified candidates in HeLa cells containing a 25% overexpression of FLAG-tagged murine Bmi1 (ectopic).  Candidates were selected which were enriched in both Bmi1 and FLAG-Bmi1 GRIP versus input.  Knockdown of 17 of these candidates was performed (vs a scramble control) and total RNA was sequenced 48 hours after knockdown.  One candidate, CAT7 was also knocked down in developing (motor) neural cells (grown from human embryonic stem cells).  We also performed ChIP targeting Polycomb proteins in the HeLa cells depleted for CAT7 or scramble controls.

overall_design: There are 2 GRIPs and their control, the input.  17 lncRNAs were selected for knockdown, and RNA was sequenced 48 hours after knockdown (n=1). 11 of these knockdowns were replicated.  3 scramble controls were also sequenced as well as mRNA, intron, and lncRNA controls (in singlicate).

first: Mridula

last: Ray

first: Ole

last: Wiskow

first: Matthew

last: King

first: Nidha

last: Ismail

first: Ayla

last: Ergun

first: Yanqun

last: Wang

first: Aaron

last: Plys

first: Chris

middle: P

last: Davis

first: Katie

last: Kathrein

first: Ruslan

last: Sadreyev

first: Mark

last: Borowsky

first: Kevin

last: Eggan

first: Leonard

last: Zon

first: Jenna

middle: L

last: Galloway


title: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma [shortRNA]

status: Public on Jul 06 2016

city: Baltimore

first: Patrick

last: Cahan

state: MD

address: 733 North Broadway, MRB 653

department: ICE and Biomedical Engineering

country: USA

institute: Johns Hopkins University School of Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: The let-7 microRNA families are tumor suppressors often deregulated in cancer, yet the underlying mechanisms of let-7 disruption remain poorly understood. Neuroblastoma is defined in part by poor prognosis associated with genetic amplification of MYCN, itself a let-7  target. The let-7 biogenesis inhibitor LIN28B  has been implicated as a critical regulator of MYCN ; however, here we show that LIN28B is dispensable for both MYCN  protein expression and growth of MYCN amplified neuroblastoma cell lines despite robust de-repression of let-7 . We further report that amplified MYCN  mRNA is a potent let-7  sponge that through exceptionally high expression defines a sub-set of self-sponging amplified competing endogenous RNA (aceRNA) and reconciles the dispensability of LIN28B . In addition, we observe frequent genomic loss of let-7 that inversely associates with MYCN- amplification, providing an explanation for common, yet unresolved amplification-independent patterns of chromosome loss. We thus propose a model whereby let-7 disruption by genetic loss, LIN28B expression, or aceRNA sponging is a unifying mechanism of neuroblastoma pathogenesis. Indeed, our data show that the majority of neuroblastomas have at least one let-7 disruption event and that genetic loss in non-MYCN amplified tumors marks decreased survival, further underscoring its importance. The inverse relationship between allelic loss and sponging of let-7 from highly expressed or amplified oncogenes may have broad implications for oncogenesis.

overall_design: Two cell lines, sampled in triplicate.

first: John

middle: T

last: Powers

first: George

middle: Q

last: Daley


title: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma [longRNA]

type: Expression profiling by high throughput sequencing

overall_design: Three cell lines, sampled in triplicate.


title: Genome-wide mapping of HIF-2α binding under normoxia in human bronchial epithelial cells (BEAS-2B)

city: Taipei

first: Meng-Chang

last: Lee

address: No.161, Sec. 6, Minquan E. Rd., Neihu Dist.,

country: Taiwan

institute: National Defense Medical Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Growing number of cancer (stem) cells and stem cells were described to accommodate constituent active HIF-2α under normoxia. Previous study of hypoxia effect may have obscured some of normoxic HIF-2α functions as hypoxia inducible features. Our interest in study of protective potential of HIFs in lung cells led us to discover pseudohypoxia functions of HIF-2α under normoxia in human bronchial epithelial cells which exhibit pluripotency related markers and features. Our study provided perspective to pseudohypoxia functions of HIF-2α via enrichment of de novo motifs. In addition to the C-TAD, the N-TAD of HIF-2α was found to contribute to targeting on these non-canonical loci. Further elucidation of pseudohypoxia mechanism could resolve its implication of malignancy or pluripotency.

overall_design: We report globally mapped binding of HIF-2α under normoxia by using high throughput sequencing

first: Chia-Yang

last: Shiau


title: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma

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

overall_design: Refer to individual Series


title: Metabolic and transcriptomic responses of leaves from European potato reference cultivars with differential tolerance to long-term drought

city: Potsdam

first: Ellen

last: Zuther

address: Am Muehlenberg 1

department: Transcript Profiling

country: Germany

institute: MPI of Molecular Plant Physiology

summary: Systems responses of mature leaves from 4 reference cultivars of a larger collection of European potato cultivars (Solanum tuberosum L.) are investigated by metabolome profiling and RNA-Sequencing. The chosen reference cultivars, Milva, Alegria, Desiree, and Saturna, vary in ascending order in regard to drought tolerance. Systems analyses are based on 3 independent field trials and 3 paralleled greenhouse trials. Robust responses across all cultivars and conditions to natural seasonal drought stress comprise proline, raffinose, galactinol, arabitol, arabinonic acid, chlorogenic acid, and 102 transcripts which consist to a high proportion of heat shock proteins and genes with signaling or regulatory functions, such as a homolog of abscisic acid receptor PYL4. Constitutive differences of the tolerant cultivars, Desiree and Saturna, compared to the sensitive cultivars include arbutin (hydroquinone-β-D-glucopyranoside), octopamine (p-hydroxyphenylethanolamine), ribitol and 248 differential transcripts. Many of these transcripts are disease related, receptor kinases, or regulatory genes, for example a homolog of the Arabidopsis FOUR LIPS MYB-regulator of stomatal cell proliferation. Functional enrichment analyses imply that heat stress is a major acclimation component of potato leaves to agronomical relevant drought stress. Enhanced leaf heat stress is a result of drought caused by loss of transpiration cooling. This effect and CO2-limitation are the main dilemmas of drought- or ABA-induced stomatal closure. Constitutive differences between tolerant and sensitive cultivars indicate partially synergistic interactions of drought and biotic stress responses. We suggest that drought tolerance of the potato reference cultivars may be caused by general resistance mechanisms which are part of previously selected pathogen tolerance.

overall_design: Transcriptome profiling by RNA-sequencing of 48 leaf samples from 4 potato cultivars grown under control or drought stress conditions in 6 independent experiments

first: Heike

last: Sprenger

first: Christina

last: Kurowsky

first: Renate

last: Horn

first: Alexander

last: Erban

first: Sylvia

last: Seddig

first: Katharina

last: Rudack

first: Axel

last: Fischer

first: Dirk

last: Walther

first: Karin

last: Köhl

middle: K

last: Hincha

first: Joachim

last: Kopka


title: An improved method for identifying biological circular RNAs

status: Public on Jul 05 2016

city: reno

first: Wei

last: Yan

state: NV

address: 1664 N. Virginia Street

department: Physiology

institute: University of Nevada

summary:
Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs
Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks.
Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases.  It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs.
Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: The wild type mouse testis RNAs were constructed NGS library by two different enzyme, then parallel sequenced in illumina Nextseq

first: Chong

last: Tang

first: Tian

last: Yu


title: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma [Let7Targets]

first: Patrick

last: Cahan

overall_design: BE(2)C:MYCN-ORF cells transfected with siRNA and miRNA mimics

first: John

last: Powers

first: George

last: Daley


title: Transcriptome of Shox2+ cells in the developing limb

city: New Orleans

first: Yiping

last: Chen

state: LA

address: 6400 Freret

department: CMB

institute: Tulane University

summary: Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.

overall_design: RNA sequencing profiling the transcriptome of Shox2+ in the developing limb

first: Wenduo

last: Ye


title: Genome-wide comparison of gene expression level between leukemia stem cells in different tissues

status: Public on Jul 07 2016

city: Aurora

first: Haobin

state: Colorado

address: 12700 East 19th Ave, Room 9122

department: Hematology

institute: University of Colorado

summary: Analysis of gene expression of leukemia stem cells (LSCs) in different tissues. The hypothesis is that the distinct niche in different tissues affects the gene expression of LSCs.

overall_design: Total RNA from LSCs in different tissues including bone marrow (BM), spleen (SPL), peripheral blood (BL), gonadal adipose tissue (AT) as well as normal counterparts for LSCs from normal BM (NBM) was isolated and subjected to RNA-seq. LSCs were derived from a blast crisis chronic myeloid leukemia (bcCML) murine model induced by co-expression of human leukemic oncogenes BCR-ABL and NUP98-HOXA9. Triplicates were generated for LSCs from each tissues.

first: Craig

last: Jordan


title: Transcriptome data used for gene annotation

city: chengdu

first: Mingzhou

last: Li

state: Sichuan

address: No.211 Huimin Road, Wenjiang District

country: China

institute: Sichuan Agricultural University

summary: To optimize the genome annotation, nine tissue and one pool RNA libraries (i.e. heart, liver, spleen, lung, kidney, muscle, fat, ovary, pool.) were constructed using the Illumina mRNA-spleeneq Prep Kit

overall_design: We sequenced nine tissues and one pool using illumina Hiseq 2500 platform

first: Li

last: Mingzhou


title: Characterization of the LFR Genomic Islet in Staphylococcus aureus CC30

city: Rochester

first: Steven

last: Gill

state: NY

address: Box 672, Elmwood Avenue

department: Microbiology and Immunology

institute: University of Rochester School of Medicine and Dentistry

summary: We report the application of RNA-Seq to assess transcriptional profiles of S. aureus CC30 strains with allelic replacements (knockouts) of two genes in the LFR genomic islet: 1) fatty acid desaturase (fad) and 2) a MocR regulator.  The overall results of our study suggest that the LFR islet enhances metabolic plasticity of the CC30 lineage which contributes to increased colonization, survival and persistence in the host.

overall_design: The background strain used for these experiments was UAMS-1. Allelic replacements of fad and MocR were made in UAMS-1. The RNA-Seq experiments compared the transcriptional profiles of UAMS-1 vs. delta fad, and UAMS-1 vs. delta MocR. Two biological replicates were used for each RNA-Seq analysis.

middle: R


title: Distinct and shared functions of ALS-associated TDP-43, FUS, and TAF15 revealed by comprehensive multi-system integrative analyses [RNA-Seq_Stability]

city: La Jolla

first: Gene

last: Yeo

state: CA

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

institute: UCSD

summary: TDP-43, FUS, and TAF15 are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We integrate CLIP-seq and RNA Bind-N-Seq technologies to discover that TAF15 binds to ~4,900 RNAs enriched for GGUA motifs. In the mouse brain, TAF15 and FUS, but not TDP-43, exhibit strikingly similar RNA binding profiles, yet they alter the expression of distinct mRNA populations upon their individual depletions. TAF15 has a minimal role in alternative splicing and instead affects RNA turnover, consistent with an enrichment of TAF15 binding sites in 3’ untranslated regions. In human stem cell-derived motor neurons, loss of both TAF15 and FUS affected mRNAs distinct from those altered by loss of either protein alone, revealing redundant roles for TAF15 and FUS in maintaining mRNA levels. Furthermore, concomitant rather than individual depletion of TAF15 and FUS more closely resembles RNA profiles of motor neurons derived from FUS R521G ALS patients or from late-stage, sporadic ALS patients. Our study reveals convergent and divergent mechanisms by which FUS, TAF15 and TDP-43 affects RNA metabolism in neurological disease.

overall_design:
RNA-seq, CLIP-seq and arrays in mouse and human against TAF15 knockdowns
This Series represents RNA-seq sample(s).

first: Katannya

last: Kapeli

first: Gabriel

last: Pratt

first: Anthony

last: Vu

first: Kasey

last: Hutt

first: Fernando

middle: J

last: Martinez

first: Balaji

last: Sundararaman

first: Ranjan

last: Batra

first: Peter

last: Freese

first: Nicole

last: Lambert

first: Stephanie

middle: C

last: Huelga

first: Seung

last: Chun

first: Tiffany

middle: Y

last: Liang

first: Jeremy

last: Chang

first: Shawn

last: Hoon

last: Donohue

first: Lily

last: Shiue

first: Jiayu

last: Zhang

first: Haining

last: Zhu

first: Franca

last: Cambi

first: Edward

last: Kasarskis

first: Manuel

last: Ares Jr

first: Christopher

middle: B

last: Burge

last: Ravits

first: Frank

last: Rigo

middle: W


title: Integrative fine-mapping of regulatory variants and mechanisms at coronary artery disease loci

city: Stanford

first: Thomas

last: Quertermous

address: 300 Pasteur Drive

department: Medicine Cardiology

institute: Stanford University

summary: Coronary artery disease (CAD) is the leading cause of mortality and morbidity driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with CAD and myocardial infarction (MI) susceptibility in multi-ethnic populations. The majority of these variants reside in non-coding regulatory regions and are co-inherited with hundreds of candidate regulatory SNPs. Herein, we use integrative genomic, epigenomic, and transcriptomic fine-mapping in human coronary artery smooth muscle cells (HCASMC) and tissues to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps we prioritize 65 candidate variants and perform allele-specific binding and expression analyses on 7 top candidates. We validate our findings in two independent cohorts of diseased human arterial expression quantitative trait loci (eQTL), which together demonstrate fundamental links between CAD associations and regulatory function in the appropriate disease context.

overall_design: We performed ATAC-seq, ChIP-seq, and RNA-seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182). For ATAC-seq and RNA-seq we performed stimulations with growth factors (TGF-B1, PDGF-BB, PDGF-DD) versus serum-free control. We conducted two biological replicates for each condition using independent donors. For ATAC-seq experiments, sequencing was completed on an Illumina Hiseq 2500, paired-end 50bp reads. For ChIP-seq we performed immunoprecipitations using H3K27ac (Abcam ab4729). We conducted two biological replicates using HCASMC from independent donors, and also did an IgG control for these studies. For RNA-seq we also conducted two replicates using HCASMC from independent donors. For both ChIP-seq and RNA-seq experiments, sequencing was completed on an Illumina HiSeq 2500, paired-end 100bp reads. We also performed ex-vivo ATAC-seq on frozen tissues (isolated media) from normal and atherosclerotic human coronary arteries, using three independent donors for each. Sequencing was also completed on an Illumina HiSeq 2500, paired end 50bp reads.

first: Clint

last: Miller

first: Milos

last: Pjanic


title: Shox2/TALE ChIP-Seq

first: Yiping

last: Chen

summary:
ChIP-Sequencing on Shox2-HA E12.5 and E13.5 Limb and Palate, as well as Pbx on E12.5 limb .

Abstract:

Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.

overall_design:
Shox2/TALE

For ChIP-Seq, the list of libraries below, including controls, were generated [listed in the format of (antibody)-target-tissue-stage]: (α-HA)-Shox2-Limb-E12.5, (α-HA)-Shox2-Limb-E13.5, (α-HA)-Shox2-Palate-E12.5, (α-HA)-Shox2-Limb/Palate-E12.5, (α-Pbx)-Pbx-Limb-E12.5, Input (control), (α-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control).

*The attached signal tracks(*.bigwig) were generated by –bdgcmp (MACS2) to filter out background signal(by filtering against the signal track obtained from (α-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control)) and subsequently convert to bigwig for analysis and visualization.

first: Wenduo

last: Ye


title: Distinct and shared functions of ALS-associated TDP-43, FUS, and TAF15 revealed by comprehensive multi-system integrative analyses [RNA-Seq_human]

first: Gene

last: Yeo

first: Katannya

last: Kapeli

first: Gabriel

last: Pratt

first: Anthony

last: Vu

first: Kasey

last: Hutt

first: Fernando

last: Martinez

first: Balaji

last: Sundararaman

first: Ranjan

last: Batra

first: Peter

last: Freese

first: Nicole

last: Lambert

first: Stephanie

last: Huelga

first: Seung

last: Chun

last: Liang

last: Chang

first: John


title: Distinct and shared molecular targets and functions of ALS-associated TDP-43, FUS, and TAF15 revealed by comprehensive multi-system integrative analyses [CLIP-Seq]

overall_design:
RNA-seq, CLIP-seq and arrays in mouse and human against TAF15 knockdowns
This Series represents CLIP-seq sample(s).

first: Tiffany

first: Jeremy

last: Donohue

first: Lily

last: Shiue

first: Jiayu

last: Zhang

first: Haining

last: Zhu

first: Franca

last: Cambi

first: Edward

last: Kasarskis

first: Manuel

last: Ares Jr

first: Christopher

last: Burge

last: Ravits

first: Frank

last: Rigo


title: Distinct and shared functions of ALS-associated TDP-43, FUS, and TAF15 revealed by comprehensive multi-system integrative analyses [RNA-Seq_mouse]

first: Gene

last: Yeo

first: Katannya

last: Kapeli

first: Gabriel

last: Pratt

first: Anthony

last: Vu

first: Kasey

last: Hutt

first: Fernando

last: Martinez

first: Balaji

last: Sundararaman

last: Batra

first: Shawn

last: Hoon


title: DNA Cytosine Hydroxymethylation Levels Are Distinct Among Peripheral Blood Leukocytes

city: Athens

first: Robert

last: Schmitz

state: GA

address: B416 Davison Life Sciences

department: Genetics

institute: University of Georgia

type: Methylation profiling by high throughput sequencing

summary: Peripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease related stress. We considered the hypothesis that the TET enzyme catalyzed hydroxymethylation of 5mC to 5hmC might vary among peripheral blood leukocytes and reflect their responsiveness to environment. Reduction in TET1 and/or TET2 activity leads to the over-proliferation of various leukocyte precursors in bone marrow and acute leukemia, yet, the role of 5mC hydroxymethylation in peripheral blood is less well studied. We developed simplified protocols to rapidly and reiteratively isolate mostly non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B) and turnover by de-methylation (TET1, 2, 3) and DNA repair (GADD45a, b, g) and in the gene-region-specific levels of DNA 5hmC (CD4 T cells >> CD14 monocytes > CD16 neutrophils > CD19 B cells > CD56 NK cells > Siglec 8 eosinophils > CD8 T cells). Taken together our results suggest a hierarchy of responsiveness among classes of leukocytes with CD4+ and CD8+ T cells and CD14 monocytes being the most distinctly potentiated for a rapid methylome response to physiological stress and disease.

overall_design: TAB-seq data on 5-hydroxymehtylcytosine (Yu, M. et al. 2012. Cell 149, 1368-1380.) was collected from seven leukocyte types (CD4+ T cells, CD8+ T cells, CD14+ monocytes, CD16+ neutrophils, CD19+ B cells, CD56+ natural killer cells, and Siglec-8+ eosinophils) reiteratively isolated from peripheral blood collected from a healthy male.


title: Distinct and shared functions of ALS-associated TDP-43, FUS, and TAF15 revealed by comprehensive multi-system integrative analyses

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Genome-wide profiling of cardiac H3K9me2 levels in response to ischemic preconditioning

status: Public on Jul 04 2016

city: Lund

first: Olof

last: Gidlof

address: Solvegatan 19

country: Sweden

institute: Lund University

summary: H3K9me2 ChIP-Seq of cardiac biopsies from the area at risk and remote myocardium of mice subjected to ischemic preconditioning.

overall_design: Mice were subjected to ischemic preconditioning (IPC) through reversible ligation of the left coronary artery or a sham procedure. The procedure consisted of 5 minutes of ischemia followed by 5 minutes of reperfusion, repeated 4 times and then followed by a 30 minute reperfusion period. Biopsies were taken from the area at risk (AAR) and remote myocardium (RM) from six IPC mice

first: Bohdan

last: Khomtchouk

first: Andrea

last: Johnstone

first: Derek

last: van Booven

first: Claes

last: Wahlestedt

first: Bernhard

last: Metzler

first: David

last: Erlinge


title: Analysis of chromosomal aberrations and recombination by allelic bias in RNA-Seq

city: Pardess hana

first: Uri

last: Weissbein

state: Please Select

address: Haalon 4

country: Israel

institute: Hebrew University of Jerusalem

summary: Genomic instability has profound effects on cellular phenotypes. Studies have shown that pluripotent cells with abnormal karyotypes may grow faster, differentiate less and become more resistance to apoptosis. Previously, we showed that microarray gene expression profiles can be utilized for the analysis of chromosomal aberrations by comparing gene expression levels between normal and aneuploid samples. Here we adopted this method for RNA-Seq data and present eSNP-Karyotyping for the detection of chromosomal aberrations, based on measuring the ratio of expression between the two alleles. We demonstrate its ability to detect chromosomal gains and losses in pluripotent cells and their derivatives, as well as meiotic recombination patterns. This method is advantageous since it does not require matched diploid samples for comparison, is less sensitive to global expression changes caused by the aberration, and utilizes already available gene expression profiles to determine chromosomal aberrations.

overall_design: RNA was extracted from different cell lines and analyzed for chromosomal stability using eSNP-Karyotyping

first: Dieter

last: Egli

first: Nissim

last: Benvenisty


title: MEIS2 is a novel oncogenic partner in AML1-ETO positive AML

city: Ulm

first: Medhanie

middle: Assmelash

last: Mulaw

address: Albert-Einstein-Allee 11

institute: University of Ulm


title: MEIS2 is a novel oncogenic partner in AML1-ETO positive AML [RNA-Seq mouse]

summary: MEIS2 collaborates with AML1-ETO in inducing acute myeloid leukemia in a murine bone marrow transplantation model

overall_design: We employed RNA-seq to assess similarities/differences among murine leukemic bone marrow samples transduced with either AML1-ETO/Meis2, AML1-ETO9a/Meis2, or AML1-ETO9a

middle: A


title: MEIS2 is a novel oncogenic partner in AML1-ETO positive AML [RNA-Seq human]

overall_design: RNA-seq to evaluate consequence of MEIS2 Knock-down on the transcriptional profle of the t(8;21) positive Kasumi-1 cell line


title: Systems level analyses establish CodY as a multifaceted metabolic regulator integrating metabolism, motility and virulence in Listeria monocytogenes [ChIP-Seq]

status: Public on Jul 03 2016

city: Tel Aviv

first: Lior

last: Lobel

state: Israel

address: Ramat Aviv

department: Molecular Microbiology

institute: Tel Aviv University

summary:
To gain knowledge on L. monocytogenes differential CodY activity, we set on analyzing CodY’s regulon in L. monocytogenes in both rich and minimal growth conditions using genome
wide sequencing techniques. Remarkably, we identified for the first time a global regulatory role for CodY when BCAAs are limited, that are similar to those within the mammalian niche. Furthermore, our data establish CodY as a central regulator that integrates metabolism, motility, stress responses and virulence in L. monocytogenes.

overall_design: Examination of CodY regulon in rich and minimal conditions.

first: Anat

last: Herskovits


title: Transcriptome comparison reveals a genetic network regulating the lower temperature limit in fish

city: Shanghai

first: Peng

last: Hu

address: Huchenghuan Road 999

institute: Shanghai Ocean University

summary: Transcriptional plasticity is a major driver of phenotypic differences between species. The lower temperature limit (LTL), namely the lower end of survival temperature, is an important trait delimiting the geographical distribution of a species, however, the genetic mechanisms are poorly understood. We investigated the inter-species transcriptional diversification in cold responses between zebrafish Danio rerio and tilapia Oreochromis niloticus, which were reared at a common temperature (28°C) but have distinct LTLs. We identified significant expressional divergence between the two species in the orthologous genes from gills when the temperature cooled to the LTL of tilapia (8°C). Five KEGG pathways were found sequentially over-represented in the zebrafish/tilapia divergently expressed genes in the duration (12 hour) of 8°C exposure, forming a signaling cascade from metabolic regulation to apoptosis via FoxO signaling. Consistently, we found differential progression of apoptosis in the gills of the two species in which zebrafish manifested a delayed and milder apoptotic phenotype than tilapia, corresponding with a lower LTL of zebrafish. We identified diverged expression in 25 apoptosis-related transcription factors between the two species which forms an interacting network with diverged factors involving the FoxO signaling and metabolic regulation. We propose a genetic network which regulates LTL in fishes.

sample_organism:
Danio rerio
Oreochromis niloticus

platform_organism:
Danio rerio
Oreochromis niloticus

overall_design: Examination of gene expressional divergence in gill between zebrafish and tilapia

first: Liangbiao

last: Chen


title: Identification of a regulatory domain controlling the Nppa-Nppb gene cluster during heart development and stress

status: Public on Jul 02 2016

city: Amsterdam

first: Vincent

middle: M.

last: Christoffels

state: N/A (NA)

address: Meibergdreef 15  K2-159

department: Medical Biology

country: Netherlands

institute: Amsterdam UMC

summary: The paralogous genes Nppa and Nppb are organized in an evolutionary conserved cluster and are a valuable model to study coregulation and regulatory landscape organization during heart development and disease. Here, we analyzed the chromatin conformation, epigenetic status and enhancer potential of sequences of the Nppa-Nppb cluster in vivo. Our data indicate that the regulatory landscape of the cluster is present within a 60 kbp domain centered around Nppb. Both promoters and several potential regulatory elements interact with each other in a similar manner in different tissues and developmental stages. The distribution of H3K27ac and the association of Pol2 across the locus changed during cardiac hypertrophy, revealing their potential involvement in stress-mediated gene regulation. In summary, the developmental regulation and stress-response of the Nppa-Nppb cluster involve the concerted action of multiple enhancers and epigenetic changes distributed across a structurally rigid regulatory domain.

overall_design: We have used 4C-seq on several viewpoints around the Nppa-Nppb gene cluster in the heart and liver samples to investigate the role of chromatin conformation on regulation of Nppa and Nppb expression during heart development and disease.

first: Irina

last: Sergeeva

first: Harmen

last: van de Werken

middle: M


title: Transcriptome profiling of self-renewing hESCs and multipotent mesoderm progenitor cells as a function of substrate stiffness

status: Public on Jul 01 2016

city: San Francisco

first: Laralynne

last: Przybyla

address: 513 Parnassus Ave.

institute: UCSF

summary: We performed RNA-sequencing on human embryonic stem cell samples grown on soft (400Pa) and stiff (60kPa) hydrogels under self-renewal and differentiation conditions

overall_design: Whole-transcriptome RNA sequencing in the conditions described

first: Valerie

last: Weaver


title: Knock-down of the methyltransferase Kmt6 reliefs H3K27me3 and results in induction of cryptic and otherwise silent secondary metabolite gene clusters in Fusarium fujikuroi [ChIP-seq]

city: Freising

first: Ulrich

last: Guldener

state: Bavaria

address: Maximus-von-Imhof-Forum 3

department: Chair of genome-oriented bioinformatics

institute: Technische Universität München

summary: We performed ChIP-seq of H3K27me3 in wild type Fusarium fujikuroi grown in synthetic ICI medium with low nitrogen conditions.

overall_design: Three replicates of F. fujikuroi wild-type strain were grown in low nitrogen. ChIP-Seq was performed with anti-H3K27me3 antibody.

first: Lena

last: Studt

first: Sarah

last: Rösler

first: Immo

last: Burkhardt

first: Birgit

last: Arndt

first: Michael

last: Freitag

first: Hans-Ulrich

last: Humpf

first: Jeroen

middle: S

last: Dickschat

first: Bettina

last: Tudzynski


title: mRNA-seq analysis of HCT116 cells following gene overexpression of IDO1 with or without Indole treatment

city: Seoul

first: Ji-Hee

last: Shin

address: 1, Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary: To investigate the effect of indole on protection against colon injury and the role of IDO1 expression, we examined the effect of indole on hIDO1-overexpressing and empty vector control HCT116 cells by global gene expression profiling.

overall_design: Examination of mRNA levels from hIDO1-overexpressing and control HCT116 cell lines treated with 1mM indole or DMF for 24 hours using two replicates each.

first: Young-Kwan

last: Lee


title: Sequential expression of Sclerotinia sclerotiorum pathogenicity factors during infection of Brassica napus as revealed by RNA-Seq analysis

city: Saskatoon

first: Dwayne

last: Hegedus

state: SK

address: 107 Science Place

country: Canada

institute: Agriculture and Agri-Food Canada

summary: RNA-seq of Sclerotinia sclerotiorum-infected B. napus leaves during a timecourse after inoculation.

sample_organism:
Brassica napus
Sclerotinia sclerotiorum

platform_organism:
Brassica napus
Sclerotinia sclerotiorum

overall_design: This is a time course experiment.  Sclerotinia hyphae were place on detached B. napus leaves, then the combined tissues were harvested at 1, 3, 6, 12, 24, and 48 hours post-innoculation.  3 biological replicates were collected for each time point, and sclerotinia from liquid medium was used as a control.

first: Shirin

last: Seifbarghi

middle: D

first: Mohammad

middle: H

last: Borhan

first: Yangdou

last: Wei

first: Cathy

last: Coutu

first: Steve

last: Robinson


title: Epigenetics and energetics in ventral hippocampus mediate rapid antidepressant action: implications for treatment-resistance

city: New York

first: Carla

last: Nasca

address: 1 Park Avenue

institute: NYU Grossman School of Medicine

summary: Although regulation of energy metabolism has been linked with multiple disorders, its role in depression and responsiveness to antidepressants is less-known. We found that an epigenetic and energetic agent, acetyl-L-carnitine (LAC, oral administration), rapidly rescued the depressive- and central and systemic metabolic-like phenotype of LAC-deficient Flinders Sensitive Line (FSL) rats. After acute stress during LAC treatment, a subset of FSL continued to respond to LAC (rFSL), whereas the other subset did not respond (nrFSL). RNAseq for the ventral dentate-gyrus (vDG), a mood-regulatory region, identified metabolic factors as key markers predisposing to depression (insulin receptors Insr, glucose transporters Glut-4 and Glut-12, the regulator of appetite Cartpt) and to LAC responsiveness (leptin receptors Lepr, metabotropic glutamate receptors-2 mGlu2, neuropeptide-Y NPY, and mineralocorticoid receptors MR). Furthermore, we found that stress-induced treatment-resistance in nrFSL shows a new gene profile, including the metabolic regulator factors Elovl7 and Cyb5r2 and the synaptic regulator NPAS4. Finally, while improving central energy regulation and exerting rapid antidepressant-like effects, LAC corrected a systemic hyperinsulinemia and hyperglycemia.tance in rFSL and failed to do that in nrFSL. These findings establish CNS energy regulation as factor to be considered for the development of better therapeutics. Agents, like LAC, which regulate metabolic factors and reduce glutamate overflow, could rapidly ameliorate depression and could also be considered for treatment of insulin-resistance in depressed subjects. The approach here serves as a model for identifying markers and underlying mechanisms of predisposition to diseases and treatment responsiveness that may be useful in translation to human behavior and psychopathology.

overall_design: Ventral dentate gyrus RNA from 3 biological replicates per group (vehicle-FRL, vehicle-FSL, LAC-treated not-responder FSL, LAC-treated responder FSL, all males) was used for library prep (TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat) and sequenced on Illumina HiSeq2500 at the Genomic Core facility at The Rockefeller University. Each sample was provided with a unique adapter and all samples were put in the same pool and run in multiple lanes to control for lane effects with a sequencing depth of about 30M (100bp, single).

first: Benedetta

last: Bigio

first: Aleksander

last: Mathe'

first: Vasco

last: Sousa

first: Danielle

last: Zelli

first: Per

last: Svenningsson

first: Bruce

last: McEwen


title: Generation of new compounds through unbalanced transcription of landomycin A cluster

city: Saarbrücken

first: Bogdan

last: Tokovenko

address: Campus C2.3

institute: Helmholtz Institute for Pharmaceutical Research Saarland

summary:
On the example of the biosynthetically exhausted landomycin A cluster we demonstrate unbalancing of gene transcription as an efficient method for the generation of new compounds.
Decoupled from the native regulators LanI and LanK, all landomycin A structural genes were set under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074.
Previously being both temporarily and quantitatively regulated, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds.
No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A.
Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host.
Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article.

overall_design: Comparison of gene expression of the H2-26 cosmid (encoding landomycin A biosynthetic genes) with H2-26-act, where an additional constitutive promoter cassette was integrated to drive biosynthetic genes transcription.

first: Maksym

last: Myronovskyi

first: Elke

last: Brötz

first: Birgit

last: Rosenkränzer

first: Niko

last: Manderscheid

first: Yuriy

last: Rebets

first: Andriy

last: Luzhetskyy


title: Knock-down of the methyltransferase Kmt6 reliefs H3K27me3 and results in induction of cryptic and otherwise silent secondary metabolite gene clusters in Fusarium fujikuroi

first: Ulrich

last: Guldener

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

overall_design: Refer to individual Series


title: RNA-seq analysis of neonatal mouse cochlear supporting cells

city: Houston

first: Andrew

last: Groves

state: TX

address: 1 Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine


title: RNA-seq analysis of neonatal mouse cochlear supporting cells [NonTreated_JM]

summary:
This study examined transcripts that are enriched in neonatal mouse cochlear supporting cells at postnatal day 1 and postnatal day 6. Supporting cells were purified by FACS sorting for GFP fluorescence from the cochleas of transgenic mice in which a BAC including the LFng locus drives the expression of GFP.
Two replicates of GFP+ supporting cells were compared with all other cochlear cell types that were GFP-. We performed this experiment at two different ages, postnatal day 1 and postnatal day 6.

overall_design: mRNA profiles of supporting cells (GFP+) and all other cochlear cell types (GFP-), two replicates each, at P1 and P6 mice were generated by deep sequencing using Illumna TruSeq.


title: RNA-seq analysis of neonatal mouse cochlear supporting cells [Treated AK]

summary:
This study examined transcripts that are enriched in neonatal mouse cochlear supporting cells at postnatal day 1 and postnatal day 6 after inhibition of the Notch signaling pathway. Cochleas from postnatal day 0 and postnatal day 5 were cultured for 24 hours in the gamma secretase inhibitor DAPT or DMSO as a vehicle control. Supporting cells were purified by FACS sorting for GFP fluorescence from the cochleas of transgenic mice in which a BAC including the LFng locus drives the expression of GFP.
Two replicates of GFP+ supporting cells were compared with all other cochlear cell types that were GFP-. We performed this experiment at two different ages, postnatal day 0+24 hours culture and postnatal day 5 + 24 hours culture.  (corresponding to P1 and P6).

overall_design: mRNA profiles of P0 and P5 supporting cells (GFP+) and all other cochlear cell types (GFP-) treated with DAPT or DMSO, two replicates each, were generated by deep sequencing using Illumna TruSeq.


title: De novo transcriptome assembly and characterization of nine tissues of Lonicera japonica to identify potential candidate genes involved in chlorogenic acid, luteolosides, and secoiridoid biosynthesis pathways

city: Chiba

first: Mami

last: Yamazaki

address: Inohana 1-8-1, Chuo-ku

department: Graduate School of Pharmaceutical Sciences

country: Japan

institute: Chiba University

summary: In this study, we performed de novo transcriptome assembly for L. japonica, representing transcripts from nine different tissues. A total of 22Gbps clean RNA-seq reads from nine tissues of L. japonica were used, resulting in 243,185 unigenes, with 99,938 unigenes annotated based on homology search using blastx against NCBI-nr protein database. Unsupervised principal component analysis and correlation studies using transcripts expression data from all nine tissues of L. japonica showed relationships between tissues explaining their association at different developmental stages. Homologs for all genes associated with chlorogenic acid, luteolin, and secoiridoid biosynthesis pathways were identified in the L. japonica transcriptome assembly. Expression of unigenes associated with chlorogenic acid were enriched in stem and leaf-2, unigenes from luteolin were enriched in stem and flowers, while unigenes from secoiridoid metabolic pathways were enriched in leaf-1 and shoot apex. Our results showed that different tissues of L. japonica are enriched with sets of unigenes associated with a specific pharmaceutically important metabolic pathways, and therefore, possess unique medicinal properties. Present study will serve as a resource for future attempts for functional characterization of enzyme coding genes within key metabolic processes.

overall_design: De novo transcriptome assembly and characterization, and transcriptome profiling  for nine tissues of Lonicera japonica

first: Kazuki

last: Saito

first: Amit

last: Rai

first: Hideyuki

last: Suzuki

first: Michimi

last: Nakamura

first: Hiroki

last: Takahashi


title: Gene expression profiling of Eed knockout and Ezh2 knockout small intestinal crypts

first: Martijn

last: Koppens

address: Plesmanlaan 121

department: Molecular Genetics

institute: NKI-AVL


title: Sunflower oil supplementation affects the expression of miR-20a-5p and miR-142-5p in the lactating bovine mammary gland

city: Jouy-en-Josas

first: Sylvain

last: Marthey

address: Domaine de Vilvert Bat 320

department: Animal Genetics

country: France

institute: INRA

summary:
Background : In lactating cow, a sunflower oil supplementation modulated the milk composition and the mammary genes expression whose mechanisms of regulations are unclear.
Results : This lipid supplementation provoke, in mammary gland, the down regulation of microRNA miR-20a-5p and miR-142-5p, which are predicted to target genes previously determined as differentially expressed including those involved in lipid metabolism.
Conclusion : Those two miRNA are good candidates to explain lipogenic genes regulations after sunflower oil supplementation.
Significance : The current study clarified the nutriregulation of miRNA in the mammary gland.

overall_design: MiRNomes obtained by NGS sequencing of mammary gland of Holstein cows at the peak of lactation received a low forage diet supplemented or not with 4% of sunflower oil were compared. MiRNA differentially expressed confirmed by RT-qPCR were related to differentially expressed genes list previously identified in the same samples.

first: Lenha

last: Mobuchon

first: Sandrine

last: Le Guillou

first: Johann

last: Laubier

first: Denis

last: Laloë

first: Fabienne

last: Le Provost

first: Christine

last: Leroux


title: MEIS2 is a novel oncogenic partner in AML1-ETO positive AML [ChIP-Seq]

first: Medhanie

last: Mulaw

overall_design: We employed ChIP-seq to assess the impact of MEIS2 knockdown on the DNA binding profile of AML1-ETO (Kasumi-1 cell line)


title: Type 1 interferons induce changes in core metabolism that are critical for immune function

first: Maxim

middle: N.

last: Artyomov

state: MO

address: 660 S. Euclid Avenue, Campus Box 8118

department: Immunology&Pathology

institute: Washington University in St.Louis

summary: Type 1 interferons (IFNs) induce complex responses that can be beneficial or deleterious, depending on context.  Greater understanding of the mechanisms of action of these cytokines could allow new therapeutic approaches.  We found that type 1 IFNs induced changes in cellular metabolism that were critical for changes in target cell function. This was apparent in plasmacytoid dendritic cells, which are specialized for type 1 IFN production, where toll-like receptor-9 (TLR9)-dependent activation was found to be dependent on increased fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) induced by autocrine signaling through the type 1 IFN receptor (IFNAR). Type 1 IFNs also induced FAO/OXPHOS in non-hematopoietic cells and were found to be responsible for increased FAO/OXPHOS in virus-infected cells.  Increased FAO/OXPHOS in response to IFNAR signaling was regulated by the nuclear receptor PPARα.  Our findings reveal PPARα/FAO/OXPHOS as potential targets to therapeutically modulate downstream effects of type 1 IFNs.

overall_design: mRNA profiles of overnight stimulated plasmacytoid dendritic cells, activated with CpG or INFa. Samples analyzed in triplicate, with HiSeq 2500 by’/ 50bpX25bp pair-end sequencing

first: Duojiao

last: Wu

first: David

middle: E

last: Sanin

first: Bart

last: Everts

first: Qiongyu

last: Chen

first: Jing

last: Qiu

first: Michael

last: Buck

first: Annette

last: Patterson

first: Amber

last: Smith

first: Chih-Hao

last: Chang

first: Zhiping

last: Liu

middle: N

first: Erika

middle: L

last: Pearce

first: Marina

last: Cella

first: Edward


title: Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions

city: Hamburg

first: Kira

last: Behrens

address: Martinistrasse 52

institute: Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology


title: Gene expression analysis to identify Runx1 target genes in GMP, MEP and   Gene expression signature of Runx1Δ/Δ lin- sca- kit+ CD105- CD16/32+ CD150+ (XMP) progenitors

summary: We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

overall_design: GMP and MEP were isolated from Runx1+/+-Tg(vav-Cre) and Runx1fl/fl-Tg(vav-Cre) mice as well as Runx1fl/fl-Tg(vav-Cre) XMP, total RNA extracted and sequenced

first: Malik

last: Alawi

first: Carol

last: Stocking


title: Transcription Factor TLX1 Controls Retinoic Acid Signaling to Ensure Spleen Development [ChIP-Seq]

city: Milano

first: Andrea

last: Brendolan

address: Via Olgettina 60

department: Experimental Oncology

country: Italy

institute: San Raffaele Scientific Institute

summary: The molecular mechanisms that underlie spleen development and congenital asplenia, a condition linked to increased risk of overwhelming infections, remain largely unknown. The transcription factor TLX1 controls cell fate specification and organ expansion during spleen development, and Tlx1 deletion causes asplenia in mice. Deregulation of TLX1 expression has recently been proposed in the pathogenesis of congenital asplenia in patients carrying mutations of the gene-encoding transcription factor SF-1. Herein, we have shown that TLX1-dependent regulation of retinoic acid (RA) metabolism is critical for spleen organogenesis. In a murine model, loss of Tlx1 during formation of the splenic anlage increased RA signaling by regulating several genes involved in RA metabolism. Uncontrolled RA activity resulted in premature differentiation of mesenchymal cells and reduced vasculogenesis of the splenic primordium. Pharmacological inhibition of RA signaling in Tlx1-deficient animals partially rescued the spleen defect. Finally, spleen growth was impaired in mice lacking either cytochrome 26B1 (Cyp26b1), which results in excess RA, or retinol dehydrogenase 10 (Rdh10), which results in RA deficiency. Together, these findings establish TLX1 as a critical regulator of RA metabolism and provide mechanistic insight into the molecular determinants of human congenital asplenia.

overall_design: We performed ChIP-sequencing for Hox11 rep1 and rep2 in eSMC untreated cells.

first: Elisa

last: Lenti

first: Diego

last: Farinello

first: Kazunari

middle: K

last: Yokoyama

first: Dmitry

last: Penkov

first: Laura

last: Castagnaro

first: Giovanni

last: Lavorgna

first: Wuputra

last: Kenly

first: Lisa

last: Sandell

first: Naomi

last: Butler Tjaden

first: Francesca

last: Bernassola

first: Nicoletta

last: Caridi

first: Anna

last: De Antoni

last: Wagner

first: Katja

last: Kozinc

first: Karen

last: Niederreither

first: Francesco

last: Blasi

first: Deigo

last: Pasini

first: Gregor

last: Majdic

last: Tonon

first: Paul

last: Trainor


title: Transcription Factor TLX1 Controls Retinoic Acid Signaling to Ensure Spleen Development

first: Andrea

last: Brendolan


title: De novo Assembly and Characterization of Swertia japonica Transcriptome for the Identification of Candidate Genes Involved in the Biosynthesis of Therapeutic Metabolites

first: Mami

last: Yamazaki

summary: Here, we performed deep transcriptome sequencing for the aerial-tissues and the roots of S. japonica, generating over 2 billion raw reads with an average length of 101 nt by using an Illumina paired-end sequencing by HiSeq2000 platform. Using a combined approach of three popular assemblers, de novo transcriptome assembly for S. japonica was obtained, yielding in 81,729 unigenes with an average length as 884bps and N50-value as 1,452bps, with 46,963 unigenes being annotated based on the sequence similarity against NCBI-nr protein database.

overall_design: Transcriptome profiling of the aerial-tissues and the roots of Swertia japonica

first: Kazuki

last: Saito

first: Amit

last: Rai

first: Hideyuki

last: Suzuki

first: Michimi

last: Nakamura

first: Hiroki

last: Takahashi


title: Genome-wide Runx1 binding sites in early hematopoietic progenitors (FDCP1)

first: Kira

last: Behrens

overall_design: ChIP analysis of Runx1 binding sites in early hematopoietic progenitors (FDCP1 and FDCP1_RUNX1-ERt2)

first: Carol

last: Stocking

first: Malik

last: Alawi


title: Gene expression profiling of Eed knockout and Ezh2 knockout small intestinal crypts [RNA-seq]

first: Martijn

last: Koppens

summary: Polycomb-mediated gene repression plays an important role in adult stem cell maintenance. We knocked out (using the inducible AhCre-LoxP system) Polycomb genes Eed and Ezh2 in the intestine for 6 weeks, after which crypts - the small intestinal stem cell zone - were harvested and RNA sequenced. We found Wnt, Notch and cell cycle pathways to be affected in Eed knockout (KO) but not Ezh2 KO crypts. Direct targets of Eed were determined by comparing this data with ChIP-sequencing.

overall_design: Small intestinal crypt mRNA profiles of 6 weeks-induced 12 weeks old Eed KO, Ezh2 KO and WT mice (all triplicates) as well as 10 days-induced Eed KO and WT organoids (duplicates) were generated by RNA sequencing over two runs and using IlluminaHiseq2000 and Hiseq2500.

first: Gergana

last: Bounova

first: Gaetano

last: Gargiulo

first: Ellen

last: Tanger

first: Hans

last: Janssen

first: Paulien

last: Cornelissen-Steijger

first: Marleen

last: Blom

first: Ji-Ying

last: Song

first: Lodewyk

middle: F

last: Wessels

first: Maarten

last: van Lohuizen


title: Gene expression profiling of Eed knockout and Ezh2 knockout small intestinal crypts [ChIP-seq]

summary: Polycomb-mediated gene repression plays an important role in adult stem cell maintenance. Direct targets of the Polycomb repressive complex PRC2 in th intestinal epithelium were revealed by performing ChIP-sequencing on crypt samples isolated from wild type murine small intestines. The resulting list of H3K27me3-enriched genes were compared with RNA-sequencing data from wild type and Eed knockout crypts.

overall_design: Crypts were isolated from wild type murine intestinal epithelium and subjected to ChIP using anti-H3K27me3 and anti-H3K27Ac antibodies, after which DNA isolated from extracted immunocomplexes was sequenced.


title: Arid3a is required for mammalian placenta development

city: Boston

first: David

middle: T

last: Scadden

state: MA

address: 185 Cambridge St

department: Center for Regenerative Medicine

institute: MGH

summary: Previous studies in the mouse indicated that Arid3a plays a critical role in the first cell fate decision required for generation of trophectoderm (TE). Here, we demonstrate that Arid3a is widely expressed during mouse and human placentation and essential for early embryonic viability. Arid3a is located within trophoblast giant cells and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Conventional Arid3a knockout embryos suffer restricted intrauterine growth with sever defects in placental structural organization. Arid3a null placentas show aberrant expression of subtype-specific markers as well as significant alteration in inflammatory response-related genes, cytokines and chemokines. We provide evidence that BMP4-mediated induction of trophoblast stem (TS)-like cells from human induced pluripotent (iPS) stem cells results in ARID3A upregulation and cytoplasmic to nuclear translocation. Overexpression of ARID3A in human iPS and BMP4-mediated TS-like cells up-regulated TE markers, whereas pluripotent markers were down-regulated. Our results indicate that the roles of Arid3a are conserved and essential for mammalian placental development through regulation of both intrinsic and extrinsic developmental programs.

overall_design: Placentas of E10.5 and E11.5 wild type (WT) and Arid3a-/- mice were generated by deep sequencing, using Illumina

first: Haley

last: Tucker


title: Dachshund ChIP-seq in Drosophila Melanogaster 3rd instar imaginal eye discs

city: Vienna

first: Jelle

last: Jacobs

address: Campus-Vienna-Biocenter 1

department: VBC

country: Austria

institute: Institute of molecular Pathology

summary: The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signalling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.

overall_design: ChIP-seq against Dachshund vs input ChIP-seq. Eye-antennal imaginal discs are dissected from Grh-GFP (Bloomington stock 42269) 3rd instar larvae and fixed with formaldehyde. Chromatin is prepared and sonicated until fragments reach an average size of 500 bp. Chromatin is immunoprecipitated with an anti-GFP Ab (ab290, Abcam) and the immunocomplexes are recovered with protein A/G magnetic beads (Millipore).

first: S

last: Aerts

first: J


title: Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation

first: Maxim

last: Artyomov

summary: Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation mechanism accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the downstream pro-inflammatory IL-1β-HIF1a axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions via its effect on succinate dehydrogenase, by inhibiting conversion of succinate to fumarate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1-/- mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, changes in mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages.

overall_design:
Experiment 1: mature WT BMDM were treated for 12h with 0.25 mM dimethyl itaconate (DI) or vehicle (Unst) and then stimulated with LPS (E. coli 0111:B4; 100 ng/ml, 4h) (DI+LPS; LPS);
Experiment 2: mature  Irg1-/- BMDM were stimulated with LPS (E. coli 0111:B4; 100 ng/ml) and murine recombinant IFNg  (50 ng/ml) for 24h.

first: Vicky

last: Lampropoulou

first: Alexey

last: Sergushichev

first: Monika

last: Bambouskova

first: Sharmila

last: Nair

first: Emma

last: Vincent

first: Ekaterina

last: Loginicheva

first: Luisa

last: Cervantes-Barragan

first: Xiucui

last: Ma

first: Stanley

middle: C

last: Huang

first: Carla

middle: J

last: Weinheimer

first: Shabaana

last: Khader

first: Gwendalyn

last: Randolph

first: Edward

last: Pearce

first: Russel

middle: G

last: Jones

first: Abhinav

last: Diwan

first: Michael

last: Diamond


title: Combined RNA-seq and Polr2a ChIP-seq Identifies Gene Targets for Transcriptional Regulation in Vasopressin-Sensitive Collecting Duct Cells

city: Bethesda

first: Pablo

last: Sandoval

state: Maryland

address: 31 Center Dr

department: NHLBI

institute: NIH

summary:
In mammals, the peptide hormone vasopressin controls renal water excretion, largely through its actions in the renal collecting duct to regulate the molecular water channel aquaporin-2 (AQP2). There are two modes of regulation:  1) short-term regulation by membrane trafficking; and 2) long-term regulation involving vasopressin-induced changes in the abundance of the aquaporin-2 protein. Defects in the long-term regulation have been implicated in multiple water balance disorders. With the objetive to identify genes that are transcriptionally regulated in response to vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq to measure all transcripts (n=9) and ChIP-seq for RNA polymerase II (Polr2a) binding along the genome (n=3). Observations were made both after 24-hr treatment with the vasopressin analog dDAVP and with vehicle.
Protocol:Confluent monolayers mpkCCD cells were washed in ice-cold 1X PBS (Ca+2/Mg+2-free) followed by cross-linking in 1.11% formaldehyde in Covaris Fixing Buffer (5.5 ml) for 5min at room temperature with gentle rocking (PN 520075, Covaris, Woburn, Massachusetts). Cross-linking was terminated by the addition of 0.3 ml of 1X Covaris Quenching Buffer with rocking. The buffer solution was aspirated and 1X ice-cold PBS was added to each plate. Cells were scraped into 15ml conical tubes, spun down (200 x g, 5 min), and washed twice with PBS. After the second wash, cell pellets were resuspended in 10 ml of 1X Covaris Lysis Buffer for 10 min at 4o C with rocking.  Nuclei were pelleted by spinning at 1,700 x g for 5 min at 4o C. Nuclear pellets was resuspended in the 1X Covaris Wash Buffer and incubated for 10 mins at 4o C with rocking. Samples were spun at 1,700 x g for 5 min at 4o C. Nuclear pellets were washed twice with the Covaris Non-ionic Shearing Buffer and spun at 1,700 x g for 5 min at 4o C. Nuclei were resuspended in Covaris Non-ionic Shearing Buffer (Maximum of 1-3x 10^7cells per ml of buffer).  Shearing.  Two 20 ul aliquots of the resuspended pellets were removed prior to shearing to be used as subsequent unsheared controls for immunoblotting and agarose gel electrophoresis. The remaining samples were transferred to AFA tubes (TC12 x 12 mm) and volumes adjusted to 1 ml (V)). Samples were sheared using a Covaris S2 SonoLAB Single system for two minutes (Duty cycle: 5%; Intensity: 4; cycle: 200). Total nuclear protein concentration of each sample in mcg/ml (R) was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).  To test shearing efficiency, a 20 ul aliquot from each tube was removed after shearing to test the efficacy of shearing. The aliquots, including the unsheared sample, were incubated with 100 ul 1X TE buffer, 10 ul 10% SDS, and RNAse A (10 ug/ul, Cell Signaling Technology, Danvers, Massachusetts) for 30 mins at 37o C. 1 ul  proteinase K (20 ug/ul) was added and the samples were incubated overnight at 65o C. The DNA was purified using DNA Clean & Concentrator columns (Catalog #: D4013, Zymo Research, Irvine, CA) and ran on an E-gel EX 2% agarose (Invitrogen, Carlsbad, CA). Target fragment sizes for chromatin immunoprecipitation were in the range of 150-700 bp in length with average fragment size of 300 bp.  Testing epitope integrity (immunoblotting).  Total protein content was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).  Samples were then diluted with sample buffer (5X Laemmli buffer) at 20% (v/v) of the entire sample volume. Proteins were resolved by SDS-PAGE (4-20% polyacrylamide gels, Criterion, Bio-Rad, Hercules, CA) and transferred electrophoretically onto nitrocellulose membranes as previously described (Hwang, 2010). Membranes were probed with anti-RNA polymerase II (Catalog #: MMS-126R, Covance). The antibody was used at a 1:500 dilution (anti-RNA polymerase II) (in Odyssey blocking buffer containing 0.1% Tween 20) overnight at 4°C. After 1-h incubation with secondary antibody (Li-Cor Biosciences 680 anti-rabbit immunoglobulin G or 800 anti-mouse immunoglobulin G; Lincoln, NE) at 1:5000 dilution, sites of antibody-antigen reaction were detected using an Odyssey infrared imager (Li-Cor).  Chromatin immunoprecipitation.  10 ul of sample equivalent to 2% input was removed prior to IP.  Chromatin Immunoprecipitation was performed using 30-90 ug of sheared chromatin in 500 uL of 1X Covaris Non-ionic Shearing Buffer with 10 μl anti-RNA polymerase II antibodies. Samples were incubated overnight at 4°C with rotation, followed by addition of ChIP Grade Protein G Magnet Beads (Catalog #: 9006, Cell Signaling Technology) and incubation for 2 h at 4o C with rotation. The beads were washed a total of four times, including three low salt washes followed by one high salt wash for 5 mins/wash at 4o C (SimpleChIP protocol #9003, Cell Signaling Technology). Immunoprecipitated chromatin was eluted from the beads with 150 ul of 1X ChIP Elution Buffer for 30 mins at 65o C in a thermomixer. To reverse crosslinks, 6 ul of 5M NaCl and 2 ul of 20 ug/ul  Proteinase K were added to the chromatin followed by incubation at 65o C overnight. DNA was purified according to SimpleChIP protocol #9003, Cell Signaling Technology.   q-PCR.    To check the success of the chromatin immunoprecipitation, input and ChIP’ed DNA samples were amplified with SYBR Green PCR Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System. Primer sets corresponding to Hox9a and actin genes were used as positive controls for both histone and RNA polymerase II IP’s. (see Supplementary Table X for primer sequences).  Library construction, amplification and sequencing.   Loaded fixed volume of total eluate. Library construction was performed on a NuGEN instrument using the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit. Libraries were then enriched via PCR. After amplification, DNA was measured.  A fixed amount of the total measured DNA was used for sequencing in an Illumina Hiseq2000 instrument to obtain single read data.  High-throughput sequencing and read mapping. Libraries for Illumina sequencing of the samples were prepared using the the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit , NuGen Mondrian SP+ (NuGEN, San Carlos, CA) . Samples were sequenced using an Illumina HiSeq2000, obtaining 1- by 50-bp reads.

overall_design: We combine RNA-seq and ChIP-seq to identify genes whose mRNA abundances change in tandem with changes in RNA polymerase II binding, as a mean to identify genes that are transcriptionally regulated in response to vasopressin.   In addition, the pattern of RNA polymerase II binding along the gene provides important information about regulatory mechanisms.   We identify two populations of genes regulated by vasopressin in mpkCCD cells, viz. 1) those with increases in transcript abundance and increases in RNA polymerase II binding throughout the gene body and; 2) genes that show increases in RNA polymerase II binding in the promotor proximal region but do not produce increases in transcript level.  The former group is restricted to 35 genes and includes Aqp2. The latter group is made up of the majority of expressed genes and is presumably due to a widespread increase in transcriptional initiation without proportionate release of pausing.

first: J’Neka

last: Claxton

first: Jae

middle: W

last: Lee

first: Fahad

last: Saeed

first: Jason

last: Hoffert

first: Mark

last: Knepper


title: Combined RNA-seq and ChIP-seq Identifies Gene Targets for Transcriptional Regulation in Vasopressin-Sensitive Collecting Duct Cells

overall_design: Refer to individual Series


title: Combined RNA-seq and ChIP-seq Identifies Gene Targets for Transcriptional Regulation in Vasopressin-Sensitive Collecting Duct Cells [RNA-seq]

summary: We sequenced mRNAs and mapping the binding of RNA polymerase 2 in collecting duct cells treat with Vasopressin or vehicle.

overall_design: Confluent mpkCCD cell monolayers were maintained in serum-free medium for 24 h prior to treatment with or without 0.1 nM dDAVP in serum-free medium for 24 h. followed  RNA polymerase 2 Chip seq.

middle: A


title: Next generation sequencing of Desulfovibrio vulgaris Hildenborough in the presence of Free Nitrous Acid stress

city: Brisbane

first: Shuhong

last: Gao

state: QLD

address: Research Road, St Lucia

department: Advanced Water Management Centre

country: Australia

institute: The University of Queensland

summary:
Purpose: Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent to a range of microorganisms. However, its antimicrobial mechanism is largely unknown. For this study Desulfovibrio vulgaris Hildenborough was selected to gain a systematic understanding of the antimicrobial mechanism of FNA.
Methods: D. vulgaris was grown under anaerobic denitrifying conditions and when FNA was added (4 μg-N/L) growth temporarily stopped. From cultures with and without added FNA, RNA was extracted and the abundance of gene transcripts was detected by sequencing. Transcripts exhibiting ≥1 or ≤-1 fold abundance change in the presence of FNA were designated as differentially expressed.
Results:The results imply that FNA exerted multiple antimicrobial effects. Growth of the culture was inhibited by FNA and significant proportions of the cells were killed during the exposure. Respiration by sulfate reduction and lactate oxidation was shut down along with ATP production when exposed to FNA. Correspondingly, decreased transcript levels of genes involved in these metabolic pathways were detected. Detoxification of FNA was evident by nitrite reduction and the genes coding both nitrite reductase (NrfA) and hydroxylamine reductase were highly up regulated. During the FNA induced energy depleted state, there was evidence that D. vulgaris lowered both ribosome activity and protein production. Conditions within the cells were more oxidizing and genes coding for proteins providing resistance to oxidative stress were up regulated. Culture thiol levels increased during the exposure, suggesting that FNA had caused protein structures to alter. Furthermore, genes for motility were up regulated possibly as an attempt to avoid FNA stress. These findings provide new insight for understanding the response of D. vulgaris to FNA exposure.
Conclusion: In this study, we performed genome-wide high throughput RNA sequencing (RNA-Seq) analysis on D. vulgaris in the absence and presence of a bacteriostatic-level of FNA (4 μg-N/L). By combining the global activated/suppressed gene profiles and detected physiological responses, we detected the microbial response to FNA exposure and revealed potential antimicrobial mechanisms of FNA on this prevalent model sulfate reducing bacteria.

overall_design: mRNA profiles of D. vulgaris Hildenborough towards FNA stress were generated by next generation sequencing in triplicate via Illumina 2000.

first: Philip

last: Bond


title: Cell fusion-mediated pluripotent reprogramming triggers ordered changes in chromatin and gene expression across the human inactive X chromosome

city: London

first: Gopuraja

last: Dharmalingam

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

department: Epigenetics section

country: United Kingdom

institute: MRC London Institute of Medical Sciences

summary: Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we used cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show a rapid and widespread loss of Xi-associated H3K27me3 and XIST in fused cells that precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-Seq analysis confirmed that Xi reactivation remained partial and showed that induction of human pluripotency-specific XACT transcripts occurred, but was rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation, and suggest a hierarchy where early events such as XIST-delocalisation, are required but are insufficient to establish stable human X reactivation.

sample_organism: Homo sapiens x Mus musculus hybrid cell line

overall_design: We performed RNA-sequencing of human fibroblast clones derived from TERT-immortalised NHDF17914 (Lonza)  before and at 5 days after fusion with mouse ESC (E14Tg2a:puroR). Two biological replicates were performed and sequenced.

first: Irene

last: Cantone


title: Characterisation of Transcriptional Changes in the Spinal Cord of the Progressive Experimental Autoimmune Encephalomyelitis ABH Mouse Model by RNA Sequencing

first: Ioanna

last: Sevastou

address: 149 Tottenham Court Road

department: Translational Research Office

institute: UCL

summary: RNA sequencing was used to explore global gene expression in the chronic relapsing and secondary progressive EAE (pEAE)  Biozzi ABH mouse model of MS. Spinal cord tissue RNA from pEAE Biozzi ABH mice and healthy littermate controls was sequenced. 2,072 genes were differentially expressed (q<0.05) from which 1,397 were significantly upregulated and 675 were significantly downregulated. This hypothesis-free investigation characterised the genomic changes that describe the pEAE mouse model.

overall_design: Examination of 3 control and 3 pEAE mice

first: I

first: G

last: Pryce

first: D

last: Baker

last: Selwood


title: RNA-seq data from murine eGFP+ relbfl/flnfkb2fl/flCg1-Cre and Cg1-Cre splenic germinal center B cells purified by fluorescent activated cell sorting.

first: Ulf

last: Klein

address: 1130 St. Nicholas Avenue

department: Herbert Irving Comprehensive Cancer Center

institute: Columbia University

summary: RNA-seq analysis of murine eGFP+ relbfl/flnfkb2fl/flCg1-Cre and Cg1-Cre splenic germinal center B cells identifies genes regulated by the transcription factors RELB and p52 (NF-kB2) in germinal center B cells.

overall_design: Germinal center B cells from 12-week old relbfl/flnfkb2fl/flCg1-Cre and Cg1-Cre littermate mice immunized with sheep red blood cells (SRBC) were isolated at day 7 after immunization by flow cytometric sorting from splenic mononuclear cells. RNA was isolated, amplified and submitted for RNA-sequencing on an Illumina HiSeq2500 instrument for 35-40 million 2x50 paired-ended reads.

first: Nilushi

last: De Silva


title: SPECtre: a spectral coherence-based classifier of actively translated transcripts from ribosome profiling sequence data

city: Ann Arbor

first: Ryan

last: Mills

state: MI

address: 100 Washtenaw Ave

department: Computational Medicine and Bioinformatics

institute: University of Michigan

summary: Active protein translation can be assessed and measured using ribosome profiling sequencing strategies. Existing approaches make use of sequence fragment length or frame occupancy to differentiate between active translation and background noise, however they do not consider additional characteristics inherent to the technology which limits their overall accuracy. Here, we present an analytical tool that models the overall tri-nucleotide periodicity of ribosomal occupancy using a classifier based on spectral coherence. Our software, SPECtre, examines the relationship of normalized ribosome profiling read coverage over a rolling series of windows along a transcript against an idealized reference signal. A comparison of SPECtre against current methods on existing and new data shows a marked improvement in accuracy for detecting active translation and exhibits overall high sensitivity at a low false discovery rate.

overall_design: Classification of actively translated transcripts in ribosome profiling data derived from human neuroblastoma SH-SY5Y cells, and data previously published derived from mouse embryonic stem cells and zebrafish embryos.

first: Sang

middle: Y

last: Chun

first: Caitlin

last: Rodriguez

first: Peter

last: Todd


title: Regulation of filamentation in the human fungal pathogen Candida tropicalis

city: Beijing

first: guanghua

last: huang

address: NO.1 West Beichen Road, Chaoyang District

institute: Institute of Microbiology

summary: The yeast-filament transition is essential for the virulence of a variety of fungi that are pathogenic to humans. N-acetylglucosamine (GlcNAc), a ubiquitous molecule in both the environment and host, is one of the most potent inducers of filamentation in Candida albicans and thermally dimorphic fungi such as Histoplasma capsulatum and Blastomyces dermatitidis. However, GlcNAc suppresses rather than promotes filamentation in Candida tropicalis, a fungal species that is closely related to C. albicans. Furthermore, we discover that glucose induces filamentous growth in C. tropicalis. Mutation and overexpression assays demonstrate that the conserved cAMP signaling pathway plays a central role in the regulation of filamentation in C. tropicalis. Activation of this pathway promotes filamentation in C. tropicalis, while inactivation of this pathway results in a serious growth defect in filamentation. By screening an overexpression library of 154 transcription factors, we have identified approximately 40 regulators of filamentous growth in C. tropicalis. Although most of the regulators (e.g., Tec1, Gat2, Nrg1, Sfl1, Sfl2, and Ash1) demonstrate a conserved role in the regulation of filamentation, similar to their homologs in C. albicans or S. cerevisiae, some of them are specific to C. tropicalis. For example, Czf1 and Efh1 repress filamentation, while Wor1, Zcf3, and Hcm1 promote filamentation in C. tropicalis. Bcr1, Aaf1, and Csr1 play a specific role in the process of GlcNAc-regulated filamentation. Our findings indicate that multiple interconnected signaling pathways are involved in the regulation of filamentation in C. tropicalis. These mechanisms have conserved and divergent features among different Candida species.

overall_design: Total RNA profiles of cells grown in Lee's glucose or Lee's GlcNAc medium.

first: Guanghua

last: Huang

first: Qiuyu

last: Zhang

first: Li

last: Tao


title: ASH1L links histone H3 lysine 36 di-methylation to leukemia

city: Palo Alto

last: Zhu

address: 265 Campus Drive, Rm. G2035

department: Pathology

institute: Stanford University

summary: The histone methyltransferases MLL and ASH1L are trithorax-group proteins that interact genetically through undefined molecular mechanisms to regulate developmental and hematopoietic gene expression.  Here we show that the lysine 36-dimethyl mark of histone H3 (H3K36me2) written by ASH1L is preferentially bound in vivo by LEDGF, an MLL-associated protein that co-localizes with MLL, ASH1L and H3K36me2 on chromatin genome wide.  Furthermore, ASH1L facilitates recruitment of LEDGF and wild type MLL proteins to chromatin at key leukemia target genes, and is a crucial regulator of MLL-dependent transcription and leukemic transformation.  Conversely KDM2A, an H3K36me2 demethylase and Polycomb-group silencing protein, antagonizes MLL-associated leukemogenesis.  Our studies illuminate the molecular mechanisms underlying epigenetic interactions wherein placement, interpretation and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a target for therapeutic intervention.

overall_design: Investigation of multiple transcription factors and histone modification marks in MV4-11 human leukemia cells.

first: Michael

last: Cleary


title: Genome-wide expression of liver and udder tissue in response to E. coli treatment in early lactation dairy cows

city: Tjele

last: Sørensen

address: Blichers Alle 1

department: Molecular Biology and Genetics

country: Denmark

institute: Aarhus University

summary: We performed a global gene-expression analysis of mammary gland and liver tissue collected from dairy cows that had been exposed to a controlled E. coli infection.

overall_design:
At time = 0, each of the periparturient dairy cows received 20-40 colony-forming units of live E. coli in one front quarter of the udder. Biopsy samples of healthy and infected udder tissue were collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent the acute phase response (APR).  A time series of liver biopsies was collected at -144, 12, 24, and 192 h relative to time of inoculation.
Hf=right forward teat
Vf=left forward teat


title: Mouse Synovial Sarcoma Models

city: Salt Lake City

first: Kevin

middle: B

last: Jones

state: Utah

address: 2000 Circle of Hope Drive, Room 4263

department: Department of Orthopaedics

institute: University of Utah

summary: Compare transcriptomes from SS18-SSX1 and SS18-SSX2 tumors

overall_design: 6 tumors analyzed, 3 from each genotype, all initiated by TATCre injection

first: Tim

last: Mosbruger


title: Histone deacetylase 3 in concert with MeCP2 and FoxO regulate autism-associated behaviors.

city: Cambridge

first: Li-Huei

last: Tsai

state: Massachusetts

department: Picower Institute for Learning and Memory

institute: MIT

summary: ChIP-Seq data of HDAC3 in mouse C57BL/6J hippocampus and RNA-Seq data of hippocampal CA1 in control and HDAC3c KO mice.

overall_design: NGS ChIP-Seq and RNA-Seq profiling of mouse hippocampus

first: Alexi

last: Nott

first: Fan

last: Gao


title: Transcriptomic and phenotypic studies of Listeria monocytogenes NmlR in stress response

city: bangkok

first: Sirirak

last: Supa-amornkul

address: 272 Payatai

department: Microbiology

country: Thailand

institute: Mahidol University

summary:
The goal of this study was to compare the transcriptome between wild type strain of Listeria monocytogenes and delete nmlR mutant strain of L. monocytogenes using NGS.
Method: Duplicate samples of rRNA depleted RNA from wild type and mutants were used to study transcriptomes by ion torrent platform.  Transcriptomes of wild type and nmlR mutant were compared by EDGE-pro program.
Result: Differential expression by EDGE-pro showed 74 genes with differential expressions between wild type and nmlR null mutant (46 genes were negatively regluated and 28 genes were positively regulated by NmlR).

overall_design: rRNA-depleted RNA samples from stationary phase wilde type and nmlR null mutant cultures were used to compare transcriptomes.  Some affected genes from RNAseq result were selected for confirmation by quantitative reverse transcriptase PCR.

first: Soraya

last: Chaturongakul

first: Wasun

last: Chantratita

first: Chutatip

last: Srichunrusamin

first: Pareena

last: Janchompoo


title: Transcription targets of ASYMMETRIC LEAVES 2 (AS2), KANADI1 (KAN), REVOLUTA (REV) and SHOOT MERISTEMLESS (STM) genes

city: Stanford

first: Samuel

last: Hokin

state: California

address: 260 Panama St

department: Plant Biology

country: USA

institute: Carnegie Institution for Science

summary: Dexamethasone applied to WT, GR-AS2, GR-KAN1, GR-REV and GR-STM seedlings and expression measured at 0, 30, 60 and 120 minutes after Dex application.

overall_design: SRA Series SRP051213 BioProject PRJNA258547. All reps are bio reps. Expression values are geometric normalized FPKM values from Tophat/Cufflinks reads and mappings.

first: M

last: Barton

first: Franklin

last: Talavera-Rauh

first: Tie

last: Liu


title: Transcriptome analysis using RNA-Seq reveals complex acid responsive mechanisms in 3-hydroxypropionic aicd producing strains of E. coli

city: Suwon-si

first: Jinsuk

middle: J.

last: Lee

address: 130 Samsung-ro

institute: Samsung Advanced Institute Technology

summary: RNA-Seq analysis was conducted at different growth phases of the cell cycle as well as various fermentation conditions. The genes encoding RNA polymerase were down-regulated in SH0012 compared to its mother strain, suggesting reprogramming of cells for survival in new environments. The micro-aerobic fermentation caused down-regulation of transporter proteins and 3-HP non-producing fermentation caused up-regulation of phage shock proteins and lipoproteins. Additionally, the clustering analyses revealed that there is a mode of temporal regulation of biological pathways during organic acid production.

overall_design: RNA transcriptome profiling of 6 samples: SH0012 (20h), SH0012 (30h), SH0012 (40h), SH0012 (no rpm change), SH0012 (no vitamin B12), SH0003 (30h, control strain)

first: In

middle: S

last: Choi


title: Identification of E2F targets in MEFs

city: Columbus

first: Lindsey

last: Kent

state: oh

address: 460 W 12th Ave

department: CCC

institute: OSU

summary: ChIP sequencing for E2F1, E2F3A and E2F3B in mouse embryonic fibroblasts

overall_design: E2F1, E2F3A and E2F3B ChIPs were performed in mouse embryonic fibroblasts stably overexpressing the proteins. ChIP DNA was processed to generate libraries and then sequenced using the Illumina platform.

first: Gustavo

last: Leone


title: Time course of androgen-regulated transcripts in LNCaP cells

city: Itabashi-ku

first: Ken-ichi

last: Takayama

address: Sakaecho

institute: Tokyo Metropolitan Institute of Gerontology

summary: Prostate cancer is the most common cancer in men and AR downstream signalings promote prostate cancer cell proliferation. To investigate the AR signaling, we performed RNA sequence analysis in AR positive prostate cancer cell line, LNCaP.

overall_design: RNA sequence analysis of androgen-regulated transcripts in prostate cancer cells

first: Satoshi

last: Inoue


title: Expression profile of wild type (WT) vs miR-155-/- in FLT3-ITD+ AML (MV4-11) cell lines

city: Salt lake city

first: Ryan

last: O'Connell

state: UT

address: 15 N Medical Dr East

summary: The expression profile in miR-155-/- FLT3-ITD+ AML is unknown. Using empty vector (EV) or two distinct miR-155 (S3 or S10) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing to determine the expression profile in these cells dependent on miR-155. We found a number of pathways dysregulated, including STAT5 activation.

overall_design: RNAseq was performed on EV or miR-155 lentiviral CRISPR-Cas9 infected MV4-11 cell lines in triplicate cultures.

middle: M

first: Jared

last: Wallace


title: Genome-wide CRISPR-Cas9 screen identifies protein-coding genes and microRNAs important in FLT3-ITD+ AML (MV4-11) cellular growth

summary: The functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to test the impact of individual microRNAs on the growth of FLT3-ITD positive leukemia cells. This approach identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote tumor cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. Our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.

overall_design: Loss-of-function CRISPR-Cas9 screen identifies genes whose loss leads to increased or decreased FLT3-ITD+ cell growth over 23 day time-course


title: Balanced E2F transcriptional output is essential for tumor suppression in the liver

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

overall_design: Refer to individual Series


title: Genome-wide CRISPR-Cas9 screen identifies functionally relevant microRNAs in AML


title: Dysregulated TLR7 signaling drives anemia via development of hemophagocytic macrophages

city: Seattle

first: Scott

last: Presnell

state: WA

address: 1201 Ninth Avenue

institute: Benaroya Research Institute

summary: The debilitating autoimmune disease Systemic Lupus Erythematosus (SLE) is closely associated with Toll-like receptor (TLR) 7 and type I interferon (IFN) activity in humans and in murine SLE-like disease. Two central manifestations of SLE affect the myeloid lineage of the immune system, myeloid expansion and anemia. Yet, whether these symptoms are linked and the role of TLR7 and/or type I IFN in these processes is unclear. Here we show that TLR7 signaling promotes cell-autonomous, phosphoinositide 3-kinase (PI3K)- and mammalian target of rapamycin (mTOR)-dependent macrophage development from the common myeloid progenitor (CMP). Strikingly, this TLR7-driven macrophage development requires and is enhanced by type I IFN. Genome-wide transcriptional profiling and functional studies demonstrated that TLR7 promoted the expression of Spic, the master regulator of splenic red pulp macrophages (RPM) and preferential development of hemophagocytic RPM-like cells from CMP in vitro. We found increased incidence of RPM-like cells in vivo in a mouse model of SLE caused by TLR7 overexpression, which correlated with decreased red blood cell (RBC) count and anemia. These findings demonstrate a mechanism by which TLR7 signaling promotes anemia that is of clinical significance in SLE, other rheumatological diseases and chronic viral infections. This work also identifies a previously unknown molecular pathway by which TLR signaling and type I IFN synergize to promote myeloid development from hematopoietic progenitors.

overall_design: CMP were sorted from the bone marrow of wild-type C57BL/6 mice, cultured with SCF+R848 or SCFr+MCSF, and CD11b+F4/80+ macrophages sorted after 5 days, n=3 per group

first: Matther

last: Buechler

first: Holly

last: Akilesh

first: Xizhang

last: Sun

first: Elizabeth

last: Whalen

first: Michael

last: Mason

first: Keith

last: Elkon

first: Jessica

last: Hamerman


title: The transcriptional landscape of mouse blood stem/progenitor cell transitions at single cell resolution

first: Evangelia

last: Diamanti

address: Wellcome Trust / MRC Building, Hills Road

department: Haematology

institute: University of Cambridge

summary: Current gene-expression databases for the haematopoietic system provide information on gene expression profiles present in bulk populations. Although informative, these studies lack the resolution that can be gained at a single-cell level. In particular, population-average data assumes homogeneity within the population and may as such obscure the ability to detect the heterogeneity of decision-making processes in individual cells. Here we report 1656 single cell transcriptomes analysed by single-cell RNA sequencing. Cells were FACS sorted on broad gates encompassing haematopoietic stem and progenitor populations (HSPCs), with index sorting data collected to permit retrospective identification of populations by surface marker expression. Our dataset thus represents the gene expression landscape of HSPCs at single-cell resolution, capturing the heterogeneity in and between cell populations. Pseudotime analysis visualized haematopoietic stem (HSC) to progenitor transitions, identified HSC as well as lineage-specific transcriptional programs, and also highlighted putative lineage branching points. To provide access to the wider scientific community, a user-friendly website was developed with intuitive search and display functionality.

overall_design: Single cell RNA sequencing of haematopoeitic stem and progenitor cells

first: Sonia

last: Nestorowa

first: Nicola

last: Wilson

first: David

first: Fiona

last: Hamey


title: Next generation sequencing identifies small RNA expression in FLT3-ITD+ AML (MV4-11) cell lines

summary: The expression level of microRNAs in FLT3-ITD+ AML is unknown. Using empty vector (EV) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing on small RNAs to determine microRNA expression level in these cells. We found a variety of evolutionarily conserved and non-conserved microRNAs expressed in our cells of interest.

overall_design: Small RNAseq on EV lentiviral CRISPR-Cas9 infected MV4-11 cell lines was performed on triplicate cultures.


title: Impact of mutations in rv2887, putative transcriptional repressor, on gene expression in Mycobacterium tuberculosis.

city: New York

first: Genomics-core-facility

last: WCM

state: NY

address: 1300 York Ave.

institute: Weill Cornell Medicine

summary: This study was aimed towards understanding the consequences of mutating rv2887, a putative transcriptional repressor, on gene expression in Mycobacterium tuberculosis. Since clones carrying mutations in this gene were found to be highly resistant to a potent mycobactericidal compound, 14, we also tested the impact of short-term exposure to this compound on gene expression in Mycobacterium tuberculosis.

overall_design: Three different strains, wild type H37Rv and the two clones resistant to compound 14, named 1A and 8A, were grown to log phase in 7H9 complete medium. The cells were diluted to an OD of 0.5 and split in to two equal parts, 1 arm received the vehicle (DMSO) and the other arm received compound 14 at a final concentration of 3.9 μM. The exposure lasted 4 hours, at which point the bacterial pellet was collected and processed for RNA extraction using protocols described previously.

first: Thulasi

last: Warrier

first: Tuo

last: Zhang

first: Carl

last: Nathan


title: Anti-Inflammatory Effects of Budesonide in Human Fetal Lung

status: Public on Jun 30 2016

city: San Francisco

first: Philip

last: Ballard

state: CA

address: 513 Parnassus St

department: Neonatology

institute: UCSF

summary:
Rationale. Lung inflammation in premature infants contributes to development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without apparent adverse effects as occur with systemic dexamethasone therapy.
Objectives. To determine effects of budesonide on differential genes expression in human fetal lung

overall_design: Methods. We prepared RNA from 3 samples of human fetal lung at 23 weeks gestation before (preculture, PC) and after 4 days culture as explants with (Bud) or without (Way) budesonide (30 nM) and performed RNAseq on the 9 samples.

first: Cheryl

last: Chapin