title: Transcriptome analysis of Aspergillus fumigatus in the presence of sugarcane bagasse

city: Piracicaba

first: Diego

middle: Mauricio Riano

last: Pachon

state: Sao Paulo

address: Av. CEntenário, 303

department: Center for Nuclear Energy in Agriculture

country: Brazil

institute: University of Sao Paulo

summary: Brazil is the world’s second largest producer of ethanol. Increasing demand for this biofuel has called for investment in new technologies. One example of such technologies is the second-generation (2G) ethanol production. Lignocellulose comprises mainly cellulose and hemicellulose, which consist of high-energy sugars that can be converted into ethanol. Aspergillus sp play an important role in the recycling of lignocellulosic biomass. These species have been investigated as a cell factory to produce industrial enzymes. To improve our understanding of the mechanisms used by Aspergillus sp during degradation of plant biomass and to identify the hydrolytic enzymes secreted by these fungi, we have analyzed the transcriptome and secretome of an Aspergillus species grown on sugarcane bagasse (SEB). A. fumigatus was cultivated in the presence of fructose or SEB. Its cellulolytic and xylanolytic activities depended on time. The maximum activities of endoglucanase and xylanase from the fungus grown on SEB were 0.0029 and 10.82 U mL-1, respectively. Characterization of the transcriptome by RNAseq technology helped to identify genes that participated in the degradation of the biomass demonstrating potential application in the process of enzymatic hydrolysis. The RNAseq data revealed that 2287 genes were differentially expressed in the fungus grown on SEB; 1181 and 1046 of these genes were up- and down-regulated, respectively. There were several CAZymes among the up-regulated genes. Most of these enzymes belonged to the GH family, which includes important hydrolytic and accessory enzymes involved in the degradation of lignocellulose. Similarly, proteomic studies showed that a total of 130 proteins existed in the fungus grown on SEB. These proteins were classified into several groups of secreted extracellular enzymes, and their functional classification demonstrated that 59% of the proteins were CAZymes such as GH45-endoglucanases, GH6 and GH7-cellobiohydrolases, GH3-β-glucosidases, GH10-xylanases, and AA9-LPMO. Data obtained by transcriptome and secretome analyses indicated that A. fumigatus produces a considerable number of CAZymes that participate in the hydrolysis of biomass. These CAZymes are valuable for the lignocellulosic bioenergy industry, provide information for future studies on the degradation of biomass, and improve understanding of the role genes and enzymes play in the degradation process.

overall_design: Two growth conditions were analysed, the only factor was the source of carbon. One condition had stem-exploded sugarcane bagasse as carbon source and the other condition had fructose. But conditions were assayed with three biological replicates.

first: Paula

middle: F

last: Gouvêa

first: Aline

last: Bernardi

first: Luis

middle: E

last: Gerolamo

first: Emerson

middle: d

last: Santos

last: Riano-Pachon

first: Sergio

last: Uyemura

first: Taisa

last: Dinamarco


title: Next Generation Sequencing assesses E2F7-regulated gene expression profiling

status: Public on Mar 18 2018

city: Bilbao

first: Ana

last: Zubiaga

address: Barrio Sarriena s/n

country: Spain

institute: University of the Basque Country

type: Expression profiling by high throughput sequencing

summary: The goal of this experiment is to analyze global gene expression profiles during the cell  cycle after acute depletion of E2F7.

overall_design: RNA was  isolated at three time-points following exit from cell cycle arrest, which represent G1/S  transition (0h), S phase (3h) and G2/M boundary (12h) of the cell cycle in cells transfected with siRNAs specific for  E2F7 (siE2F7) or with non-target control siRNAs (siNT)

first: Jone

last: Mitxelena

first: Aintzane

last: Apraiz

first: Jon

last: Vallejo-Rodríguez

first: Asier

last: Fullaondo

first: Mónica

last: Alvarez-Fernánde

first: Marcos

last: Malumbres

middle: M


title: Genome-wide occupancy of LSD1 in differentiating C2C12 myoblasts.

status: Public on Mar 19 2018

city: Kumamoto

first: Kotaro

last: Anan

state: Kumamoto

address: 2-2-1, Honjo, Chuo-ku

department: Department of Medicall Cell Biology

country: Japan

institute: Kumamoto University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ChIP-seq analysis of LSD1 in C2C12 cells. We found that LSD1 directly regulates the expression of fiber and metabolism genes during myogenesis. Results provide insight into the molecular mechanisms  of myogenesis.

overall_design: Examination of LSD1/DNA interaction in C2C12 myoblasts 48 h post-differentiation.

first: Shinjiro

last: Hino

first: Mitsuyoshi

last: Nakao


title: Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function

city: Bethesda

first: Kai

last: Ge

state: MD

address: 50 South Dr Rm 4154

department: NIDDK

country: USA

institute: NIH

summary: Histone tails are post-translationally modified at multiple sites, including Lys36 on histone H3 (H3K36). The H3K36 methylation has been shown to associate with the transcription of active euchromatin, alternative splicing, DNA repair and recombination. However, the role of H3K36 methylation during cell differentiation is still obscure.Previous investigations found that a site specific Lys-to-Met mutation on histones can serve as an inhibitor of this site specific methyltransferases to repress global methylation on that lysine of histones. In this study we usedH3K36 Lys-to-Met mutant (H3K36M) as a tool to investigaterole of H3K36 methylation in our cell differentiation system. Expression of H3K36M repressed global H3K36 methylation but increased H3K27me3 as previously reported. On our cell differentiation system, H3K36M suppressed adipogenesis and myogenesis. Our pioneer RNA-seqstudy further showed that H3K36M repressed the expression of master regulator genes. Here, we did ChIP-seq of several histone modifications to further explore the changes on the epigenome by expression of H3K36M. Interestingly, on some important master regulator genes loci, the repressive marker H3K27me3 increased significantly, correlated with the repression on their expression. The H3K36M decreases global H3K36 di- and tri-methylation. To clarify which H3K36 methyltransferase is important for cell differentiation, we knocked down H3K36 di-methyltransferases Nsd1 and Nsd2, H3K36 tri-methyltransferase Setd2 separately. Nsd2 knockdown, but not Nsd1 or Setd2,can phenocopythe adipogenesis defect in H3K36M expressed cells.Comparing in RNA-seq results, Nsd2 knockdown cells showed similar gene expression profile with H3K36M expressed cells in adipogenesis. These suggest that Nsd2-mediated H3K36me2 plays an important role in adipogenesis.To study the role of H3K36 methylation in vivo, we generated an aP2 promoter driven H3K36M expressed transgenic mouse (Tg), to express H3K36M specifically in adipose tissue. The Tg mice showedsignificant dysfunction in both white and brown adipose tissues. Their fat tissues gene expression profile changed significantly. All of these indicate that H3K36 methylation is important for adipose tissue developmentin vivo.Together, our comprehensive studies provide novel insights into dynamics of H3K36 methylation and its important role in transcriptional regulation of cell differentiation and mouse fat tissue development.

overall_design: Expression profiling by RNA-seq at 3 time points (D0, D2, D7), and DMSO or Rosi stimulation. Profiling of histone modifications (H3K36me2, H3K27me3, H3K4me1, H3K27ac) by ChIP-seq at two time points (D0, D2) during adipogenesis of brown preadipocytes in culture.

first: Lenan

last: Zhuang

first: Young-Kwon

last: Park

first: Binbin

last: Lai

first: Ji-Eun

last: Lee

first: Young-Hoon

last: Jang


title: Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding

status: Public on Mar 17 2018

city: Stanford

first: Polly

middle: Morrell

last: Fordyce

state: CA

address: 443 Via Ortega

department: Genetics, Bioengineering

institute: Stanford University

summary: Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF-DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and shed light on how transcriptional networks are rewired throughout evolution. Here, we present a novel sequencing-based TF binding assay and analysis pipeline (BET-seq, for Binding Energy Topography by sequencing) capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target for two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NN) and biophysical models trained on these data, we establish that dinucleotide specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more non-additivity than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest-affinity sites.

overall_design: Examination of S. cerevisiae transcription factors, Pho4p (4 replicates) and Cbf1p (3 replicates), interacting with synthetic duplex DNA by BET-seq.


title: Polycomb repressive complex 1 regulates the nucleosome landscape but not accessibility at target genes [ATAC-seq]

city: London

first: Hamish

middle: W

last: King

address: 4 Newark St

department: Blizard Institute

country: United Kingdom

institute: Queen Mary University of London

summary: Polycomb repressive complexes 1 and 2 (PRC1/2) act to limit transcriptional activity at gene promoters. One of the mechanisms by which they are proposed to achieve this is through the compaction of nucleosomal chromatin. However, this model is based largely upon in vitro studies and has yet to be explored sufficiently in living cells. We therefore characterised the chromatin accessibility of Polycomb-occupied gene promoters using assay for transposase accessible chromatin (ATAC-seq). Although Polycomb-occupied promoters exist in a less accessible state compared to Polycomb-free promoters, we were surprised to observe that deletion of either PRC1, PRC2 or both PRC1/2 did not result in any increases in chromatin accessibility. This was in contrast to widespread increases in the transcriptional activity of Polycomb target genes. Together with experiments examining chromatin accessibility in the context of RNA polymerase II inhibition, we therefore demonstrate an apparent uncoupling of the chromatin accessibility and transcriptional activity of gene promoters.

overall_design: Mouse embryonic stem cells with conditional (RING1Bfl/fl and EED cKO) or constitutive mutations (RING1Bfl/fl;EED-/-) for PRC1 and/or PRC2 were profiled for chromatin accessibility using ATAC-seq. Transcription was inhibited using the triptolide treatment to assess the contribution of transcriptional activity towards chromatin accessibility.

first: Robert

middle: J

last: Klose


title: Cooperative epigenetic remodeling by TET2 loss and NRAS mutation drives myeloid transformation and MEK inhibitor sensitivity [bisulfite-Seq]

city: New York

first: Francine

middle: E.

last: Garrett-Bakelman

state: NY

address: 413 East 69th street, BRB 1426

department: Medicine

institute: Weill Cornell Medicine

type: Methylation profiling by high throughput sequencing

summary: Recent studies using next-generation sequencing technology have uncovered mutational landscapes of various myeloid malignancies (Cancer Genome Atlas Research Network, 2013; Yoshida et al., 2011). These genetic data revealed novel classes of mutations that commonly occur in patients with myeloid malignancies, including epigenetic regulators and spliceosomal genes. In addition, co-occurrence and mutual exclusivity of these disease alleles suggest convergent cooperative roles or common biological effects of specific alleles in myeloid leukemogenesis (Shih et al., 2012).    Somatic deletions and loss-of-function mutations in TET2, a member of the TET family which regulates DNA methylation, were identified in 10-20% of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) patients, 12-24% of acute myeloid leukemia (AML) patients and 40-50% of chronic myelomonocytic leukemia (CMML) patients (Abdel-Wahab et al., 2009; Delhommeau et al., 2009; Jankowska et al., 2009; Langemeijer et al., 2009). Analysis carried out in large AML cohorts has shown that TET2 mutations are associated with adverse outcome in patients with intermediate-risk, cytogenetically normal AML (Patel et al., 2012). TET proteins (TET1-3) are Fe(II)- and a-ketoglutarate-dependent enzymes that catalyze the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) leading to DNA demethylation (Guo et al., 2011; Tahiliani et al., 2009). Consistent with this notion, TET2 mutant AML patient samples show decreased 5-hmC levels and hypermethylation phenotype (Figueroa et al., 2010; Ko et al., 2010). Oncometabolite 2-hydroxyglutarate produced by IDH1/2 mutations inhibits TET2 function (Figueroa et al., 2010). Moreover, WT1 recruits TET2 to regulate its target gene expression (Wang et al., 2015) and WT1 mutations lead to reduced TET2 function and decreased 5-hmC levels (Rampal et al., 2014). Together, IDH1/2 mutations and WT1 mutations share, at least partially, common epigenetic pathogenesis in AML as TET2 mutations through altered DNA hydroxymethylation.    A number of studies have shown that TET2 is a key regulator of hematopoietic stem cell (HSC) self-renewal and myeloid differentiation. Conditional loss of Tet2 in mouse hematopoietic compartment leads to expansion of hematopoietic stem/progenitor cells (HSPCs, LSK, lin- Sca1+ cKit+) and enhanced repopulating capacity in vivo (Li et al., 2011; Moran-Crusio et al., 2011; Quivoron et al., 2011). In addition, Tet2-mutant mice show myeloproliferation in vivo and TET2-silenced human cord blood cells show skewed differentiation towards CD14+ myelomonocytic lineage in vitro (Pronier et al., 2011), which is compatible with a high frequency of TET2 mutations in CMML patients. More recently, mutations in TET2 and in other epigenetic regulators, such as DNMT3A and ASXL1, are reported in individuals with clonal hematopoiesis without hematological malignancies (Busque et al., 2012; Xie et al., 2014). Analysis of AML and patients with nonhematologic malignancies suggest mutations in TET2 and in other epigenetic modifiers may represent early premalignant events that cause clonal hematopoietic expansion (Genovese et al., 2014; Jaiswal et al., 2014; Jan et al., 2012). These data indicate that TET2 inactivation contributes to selective clonal advantages in early leukemia initiation but requires additional genetic events to induce myeloid transformation.    Ras proteins are small GTPases that cycle between active guanosine triphosphate (GTP)-bound (Ras-GTP) and inactive guanosine diphosphate (GDP)-bound (Ras-GDP) conformations (Schubbert et al., 2007). Ras-GTP binds to and activates downstream signaling effectors, such as Raf and phosphoinositide 3-kinase (PI3K), thereby transducing signals from activated growth factor receptors to the nucleus. Thus, Ras-mediated signal transduction critically regulates fundamental cellular behaviors, including proliferation, differentiation and survival (Schubbert et al., 2007). The three cellular Ras genes encode 4 highly homologous proteins, Hras, Kras4A, Kras4B and Nras, that share conserved effector binding domains and the P-loop, which binds the g-phosphate of GTP (Zhou et al., 2016). Cancer-associated RAS mutations encode proteins that accumulate in the active GTP-bound conformation due to defective intrinsic hydrolysis and resistance to guanosine triphosphatase-activating proteins (Schubbert et al., 2007). Among these isoforms, NRAS is the most common target of oncogenic signaling mutations in myeloid leukemia, including 10-15% of AML and CMML cases (Bacher et al., 2006; Ball et al., 2016). Moreover, recent study identified NRAS as one of the somatic gene mutations with adverse prognostic relevance in CMML (Elena et al., 2016). Consistent with human data, mice with hematopoietic tissue specific conditional NrasG12D allele develop fatal myeloproliferative disease in vivo (Li et al., 2011). These data underscore critical role of oncogenic NRAS mutations in myeloid leukemogenesis.    Recent studies have shown that mutations in epigenetic modifiers and signaling factors commonly co-occur in AML, including the co-occurrence of TET2 mutations and NRAS mutations (Papaemmanuil et al., 2016; Patel et al., 2012). Analysis of the clonal architecture in CMML patients suggests TET2/NRAS double-mutant clones expand both within stable disease time course and in relapse after allogeneic stem cell transplantation (Itzykson et al., 2013). These evidences imply TET2 and NRAS mutation likely function together in myeloid transformation. Although previous study has shown that knockdown of Tet2 does not accelerate NrasG12D induced transplant myeloproliferative disease model (Chang et al., 2014), to date there have been no studies of the mechanistic impact caused by the combination of these high risk genetic lesions using physiologic in vivo model. Moreover, how the combination of myeloid leukemia-associated disease alleles affect sensitivity to targeted therapy has not been elucidated. Here we investigate loss of Tet2 together with NrasG12D in CMML development and specifically how they interact to affect sensitivity to targeted therapy.

overall_design: We utilized hematopoietic-tissue specific conditional Tet2/Nras double-mutant mice for our study and examined methylome of FACS-sorted LSK populations from WT, Tet2(-/-), Nras(G12D), non-leukemic Tet2(-/-) Nras(G12D), and leukemic Tet2(-/-) Nras(G12D) animals at 6 months of age. DNA was extracted using All Prep micro kit (Qiagen).

first: Hiroyoshi

last: Kunimoto

first: Cem

last: Meydan

first: Caroline

last: Sheridan

first: Tak

first: Yaseswini

last: Neelamraju

first: Ari

last: Melnick

first: Ross

middle: L

last: Levine


title: Polycomb repressive complex 1 defines the nucleosome landscape but not accessibility at target genes

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

overall_design: Refer to individual Series


title: Cooperative epigenetic remodeling by TET2 loss and NRAS mutation drives myeloid transformation and MEK inhibitor sensitivity


title: Gene expression alterations of pancreatic intraepithlial neoplasia

city: Baltimore

first: Michael

last: Ayars

address: 1550 Orleans St

institute: Johns Hopkins School of Medicine

summary: Gene expression data from the RNA sequencing of laser microdissected pancreatic intraepithelial neoplasia and pancreatic normal ducts.

overall_design: All RNA samples were isolated from laser microdissected tissues.  Samples were prepared in two sets.  13 samples (1 normal duct, 2 PanIN-1s, 3 PanIN-2s, and 7 PanIN-3s) were sequenced on an ABI SOLiD 5500 instrument.  8 samples (2 normal duct, 2 PanIN-1s, 2 PanIN-2s, and 2 PanIN-3s) were sequenced on an Illumina HiSeq instrument.  Datasets were combined using ComBat batch removal.

first: Eileen

last: O'Sullivan

last: Goggins


title: RNA-sequencing of neonatal monocyte responses to E. coli and S. epidermidis

status: Public on Mar 16 2018

city: Crawley, Perth

first: Emma

last: de Jong

state: WA

address: 35 Stirling Hwy

country: Australia

institute: University of Western Australia

summary: Newborns, particularly those born prematurely, are extremely vulnerable to sepsis and this has been attributed to ‘immature’ innate monocyte defences. Predominant pathogens include Escherichia. coli and Staphylococcus. epidermidis but no studies have assessed global transcriptional responses of neonatal monocytes to live sepsis-causing bacteria. Here, we aimed to identify and characterise the common and pathogen-specific, neonatal monocyte transcriptional responses to E. coli and S. epidermidis to better understand early life innate immune responses. RNA-sequencing was performed on purified cord blood monocytes from very preterm (<32 weeks gestational age, GA) and term infants (37-40 weeks GA) following standardised challenge with live S. epidermidis or E. coli.

overall_design: 43 samples in total from 4 term infants, 5 preterm infants exposed to chorioamnionitis and 6 preterm infants without exposure to chorioamnionitis. Each individual has an unstimulated monocyte sample, and a E. coli- or S. epidermidis-stimulated monocyte sample (3 samples per individual) except for two individuals (preterm infants without exposure to chorioamnionitis) which only have an unstimulated monocyte sample and one of either E. coli- or S. epidermidis-stimulated mnocytes.

first: David

middle: G

last: Hancock

first: Julie

last: Hibbert

first: Christine

last: Wells

first: Peter

last: Richmond

first: Karen

last: Simmer

last: Burgner

first: Tobias

last: Strunk

first: Andrew

last: Currie


title: Cooperative epigenetic remodeling by TET2 loss and NRAS mutation drives myeloid transformation and MEK inhibitor sensitivity [RNA-Seq]

first: Francine

last: Garrett-Bakelman

first: Hiroyoshi

last: Kunimoto

last: Meydan

last: Lee


city: Boulder

first: Glen

last: Bjerke

state: CO

address: 347 UCB

department: MCDB

institute: University of Colorado

summary:
The objective of this study was to identify miR-200 family member target sites.  Of note, CLEAR-CLIP captures all miRNAs, not one family specifically.  So data for all miRNAs is included.
CLEAR-CLIP on murine keratinocytes grown in culture.  Keratinocytes were either wild type, miR-200 family double knock out or miR-200b/miR-200a/miR-429 cluster inducible.

overall_design: This study includes two sets of samples.  One set is 6 replicates of control cells and 3 replicates of miR-200 DKO cells.  The other set is 3 replicates of control cells, 3 replicates of miR-200 DKO cells and 3 replicates of miR-200 inducible cells.

first: Jaimee

last: Hoefert

first: Rui

last: Yi


title: RNA-seq on biliary epithelial cells (BECs) of Klf5 LKO mice upon DDC injury

city: Mishima

first: Hajime

last: Okada

state: Shizuoka

address: Yata 1111

institute: National Institute of Genetics

summary: In other to assess functional involvement of Klf5 in DR regulation, we made liver-specific Klf5 knockout mice. Ductular reaction upon cholestatic liver injury is severely suppressed in these mice. We conducted RNA-seq analysis on the BECs from control mice and Klf5 LKO mice upon DDC injury to further elucidate the Klf5 functions.

overall_design: Single-end RNA-seq of total RNAs extracted from BECs of Klf5 LKO mice upon DDC injury for 1wk


title: Polycomb repressive complex 1 regulates the nucleosome landscape but not accessibility at target genes [RNA-seq]

first: Hamish

last: King

overall_design: Mouse embryonic stem cells with conditional (RING1Bfl/fl) or constitutive mutations (RING1Bfl/fl;EED-/-) for PRC1 and/or PRC2 were profiled for gene expression using nuclear RNA-seq.

first: Robert

last: Klose


title: The gut microbiome drives distinct methylome and transcriptome changes in intestinal epithelial cells during postnatal development

city: Kiel

first: Wei-Hung

last: Pan

address: Rosalind-Franklin-Straße 12

department: Institute of clinical molecular biology

country: Germany

institute: Christian-Albrechts-Universität zu Kiel

summary: We investigate the microbial effects on DNA methylation and the transcriptome of intestinal epithelial cells (IECs) during postnatal development

overall_design:
C57Bl6/N female littermate mice were maintained under standard specific pathogen free or GF conditions in the laboratory for experimental biomedicine at University of Gothenburg. Mice were kept under a 12-h light cycle and fed autoclaved chow diet ad libitum (Labdiet, St Louis, MO, USA). Mice were sacrificed at three different stages: 1, 4 and between 12 to 16 weeks of age. Mice were killed by cervical dislocation and the small intestine removed for isolation of IECs. All animal protocols were approved by the Gothenburg Animal Ethics Committee.

We collected IECs from the small intestine of 1-, 4- and 12 to 16-week-old mice, raised either in the presence or absence of a microbiota.

first: Philip

last: Rosenstiel

first: Felix

last: Sommer


title: The thioredoxin-1 system is essential for fueling DNA synthesis during T cell metabolic reprogramming and proliferation

status: Public on Mar 15 2018

city: Zürich

first: Manfred

last: Kopf

address: Otto-Stern-Weg 7

department: Biologie

country: Switzerland

institute: ETH Zürich

summary: The thioredoxin-1 (Trx1) system is a key player of the cellular redox balance and a sensor of energy and glucose metabolism. Here we report critical c-Myc-dependent activation of the Trx1 system during glucose-driven T cell expansion during development and immune responses but endogenous Trx1-repression during quiescence. Thioredoxin-reductase-1 (Txnrd1)-deletion in CD4-CD8- thymocytes prevented their expansion, while deletion in CD4+CD8+ thymocytes did not affect further maturation and peripheral homeostasis of T-cells. Moreover, B cell development was Txnrd1-independent. Txnrd1 was critical for expansion of activated T cells. Unbiased metabolomics revealed that Txnrd1 is necessary for donating reducing equivalent to ribonucleotide reductase (RNR) at the last step of nucleotide biosynthesis. Impaired availability of 2’-deoxyribonucleotides induced the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal role of the Trx1 system in metabolic reprograming of thymic and peripheral T cells and provide a rationale for targeting of Txnrd1 in T-cell leukemia.

overall_design: We compared naive and SEB-stimulated WT and Txnrd1-deficient CD8+ T cells. In total we have 4 conditions (WT & KO, and naive & SEB-stimulated). Each condition has 2 biological replicates.

first: Jonathan

last: Muri


title: Discovering the interactions between circRNAs and RNA-binding proteins from RNA-seq and CLIP-seq data

city: Guangzhou

first: Jian-Hua

last: Yang

address: No. 135, Xingang Xi Road

department: School of Life Sciences

country: China

institute: Sun Yat-sen University

summary: Although tens of thousands of circular RNAs (circRNAs) have been identified in mammalian genomes, only a few have been functionally characterized. Here, we report a new approach, circScan, to identify regulatory interactions between circRNAs and RNA-binding proteins (RBPs). Our method identifies back-splicing reads from crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq) data. Using our method, we systematically scanned ~1,500 CLIP-seq datasets and identified ~12,540 and ~1,090 novel circRNA-RBP interactions in the human and mouse genomes, respectively, including all known interactions between circRNAs and Argonaute (AGO) proteins. Furthermore, more than twenty novel interactions were experimentally confirmed by RNA immunoprecipitation combined with quantitative PCR (RIP-qPCR). Importantly, we determined that natural circRNAs interact with the cap-independent translation factors eukaryotic initiation factor 3 (eIF3) and N6-methyladenosine (m6A), thus suggesting that those circRNAs may be translated into proteins. These findings demonstrate that circRNAs are regulated by various RBPs and therefore may play important roles in diverse biological processes.

overall_design: Discovery of circular RNAs in 2 cell types.

first: Bin

last: Li


title: IGF2BP proteins Enhance mRNA stability

city: Monrovia

first: Jianjun

last: Chen

department: Systems Biology

institute: Beckman Research Institute of City of Hope

summary: To evaluate the effect of IGF2BPs on mRNA stability and gene expression output, we conducted RNA-seq in individual IGF2BP knockdown and control HepG2 cells with or without actinomycin D treatment. Our RNA-seq and RNA stability profiling revealed that IGF2BPs were involved in RNA stability regulation and contributed to the stabilization of the transcriptome.

overall_design: HepG2 cells were infected with individual lentiviral IGF2BP shRNA and non-specific control (shNS), and selected by puromycin to generate stable knockdown lines. We treated HepG2 cells with actinomycin D to inhibit transcription and collected cells at indicated time points (i.e., 0h, 1h, 3h, 6h). The total RNA was extracted by miRNeasy Kit (Qiagen) and sequenced by Illumina.  For IGF2BP-dependent gene expression, untreated cells (i.e., 0h samples) were sequenced in triplicate and analyzed. For RNA stability profiling, RNA half-life was calculated by comparing the gene expression at 1, 3, 6 hours with actinomycin treatment to that in un-treated samples, with two biological replicates for each group.

first: Huilin

last: Huang

first: Hengyou

last: Weng

first: Wenju

last: Sun

first: Jianhua


title: Novel transcriptional networks regulated by CLOCK in human neurons

city: Dallas

first: Genevieve

last: Konopka

state: TX

address: 5323 Harry Hines Blvd.

department: Neuroscience

institute: UT Southwestern Medical Center

summary: The molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to human cognition and disease. In this study, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin-immunoprecipitation sequencing for endogenous CLOCK in adult neocortex and RNA-sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts co-expressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks are driven by hub genes with human-specific patterns of expression. Thus, these data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function.

overall_design: We carried out RNA-sequencing (RNA-seq) and ATAC-sequencing (ATAC-seq) in differentiated human neural progenitor cells and ChIP-sequencing (ChIP-seq) in both differentiated human neural progenitor cells and BA10 and BA40 of adult human neocortex. For the Chip-seq, five replicates were used for each of the two Brodmann areas (BA10 and BA40) of adult human frozen brain tissue (i.e. the five replicates of BA10 and BA40 came from the same five individuals). For the RNA-seq, three independent replicates of control and CLOCK knockdown samples were used each time point analyzed. For the ATAC-seq, three independent replicates of control and CLOCK knockdown samples were used.

first: Miles

last: Fontenot

first: Stefano

last: Berto

first: Gordon

last: Werthmann

first: Noriyoshi

last: Usui

first: Kelly

last: Gleason

first: Carol

last: Tamminga

first: Joseph

last: Takahashi


title: PU.1 represses and activates gene expression in early T cells by redirection of transcription factor ensembles

status: Public on Mar 14 2018

city: Isehara

first: Hiroyuki

last: Hosokawa

state: Kanagawa

address: 143 Shimokasuya

department: Department of Immunology

institute: Tokai University School of Medicine


title: Adaptation Versus Senescence In Rice: Early Signalling Events Play A Role In Inducing Specificity And Tolerance To Salinity

city: PADOVA

last: Toppo

address: viale G. Colombo, 3

department: Molecular Medicine

country: Italy

institute: University of Padova

summary: Salinity tolerance is a complex trait and, despite many efforts to obtain rice plants resistant to salt, few results have been achieved since a deeper understanding of the tolerance mechanisms is still needed. We used imaging of photosynthetic parameters, ion analysis and transcriptomic approaches to unveil differences between two rice varieties differing in salt sensitivity. Moreover, we analysed H2O2 production in roots, using a fluorescent probe, and the ensuing gene regulation. Transcriptomic analyses conducted in tolerant plants supported the set-up of an adaptive program consisting of allocating sodium preferentially to roots, restricting it to the oldest leaves and activating regulatory mechanisms of photosynthesis in new leaves. As a consequence, plants resumed growth even under prolonged salt stress. By contrast, in the susceptible variety, RNA profiling unveiled a mis-targeted response, leading to senescence and cell death. In roots of tolerant plants, an increase in H2O2 was observed as early as 5 minutes after treatment. Consequently, the expression of genes involved in perception, signal transduction and response to salt were induced at earlier times when compared to susceptible plant roots. Our results demonstrate that a prompt H2O2 signalling in roots participates to a coordinated response resulting in adaptation instead of senescence in salt treated rice plants.

overall_design: Samples were collected after 3 days of treatment with 100 mM NaCl. Roots and leaves were collected separately from untreated and treated plants. Each sample was made of 6 pooled plants. Three biological replicates were performed.

first: Elide

last: Formentin

first: Paolo

last: Fontana

first: Enrico

last: Lavezzo


title: Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

first: W. Lee

last: Kraus

summary: Extracellular signaling and nutrient availability are major factors for cell fate decision. Responds to extracellular information requires metabolic alterations and differential gene expression. However, how cells integrate extracellular signals (e.g. hormones) and cellular metabolic status to coordinate transcriptional outcome is poorly understood. We hypothesized that fluctuations in nuclear nicotinamide adenine dinucleotide (NAD+)  levels act as a signal to integrate cellular glucose metabolism and transcription program during adipocyte differentiation. To test this hypothesis, we performed RNA-seq on control, Nmnat1 and Parp1 knockdown 3T3-L1 cells during various time point of differentiation.

overall_design: Using RNA-seq over a time course of DMI induced differention of 3T3-L1 preadipocytes expressing control or Nmnat1 or Parp1 shRNA.

first: Keun

last: Ryu

first: Tulip

last: Nandu

first: W


title: Recognition of RNA N6-methyadenosine by IGF2BP proteins Enhances mRNA Stability

first: Jianjun

last: Chen


title: Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence

city: Piracicaba

first: Diego

last: Pachon

address: Av. CEntenário, 303

institute: University of Sao Paulo

summary: Iron restriction imposed by mammalian hosts during an infection is a common mechanism of defense to reduce or avoid the pathogen infection. Iron is essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis (IA), a disease that typically manifests in immunocompromised patients. A. fumigatus has two high affinity mechanisms of iron acquisition during infection: reductive iron assimilation (RIA) and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. LC-MS under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A (PPA), fumagillin, fumiquinazoline A, TAFC, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production and pathogenicity in A. fumigatus.

overall_design: Time series with three time points i.e., 0, 1 and 2 h. With Wild Type and the Delta Afu2g03950 mutant, grown in iron starvation conditions. Three biological replicates for each collection point. BioProject: PRJNA381768

first: Adriana

last: Oliveira Manfiolli

first: Patrícia

last: Alves de Castro

first: Stephen

last: Dolan

first: Sean

last: Doyle

last: Riaño Pachón

first: Mevlüt

last: Ulaş

first: Luke

last: Noble

first: Derek

last: Mattern

first: Vito

last: Valiante

first: Ozgur

last: Bayram

first: Gustavo

middle: H

last: Goldman


title: Transcriptome-wide identification of IGF2BP binding RNAs by RIP-seq.

summary: We identified the mRNA targets of the insulin-like growth factor-2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) by RNA immunoprecipitation and sequencing (RIP-seq). HEK293T cells transfected with Flag-tagged IGF2BP1/2/3 plasmids were expanded and UV-crosslinked before harvest. We performed RIP of individual IGF2BP using anti-Flag antibody from nuclear extractions, and identified the associated mRNAs by next generation sequencing. More than 5000 transcripts, including protein coding and non-coding transcripts, were identified from each RIP-seq sample.

overall_design: RIP-seq in HEK293T cells with Flag-tagged IGF2BP1/2/3 overexpression.

first: Huilin

last: Huang

first: Hengyou

last: Weng

first: Wenju

last: Sun

first: Jianhua

last: Yang


title: Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

city: Emeryville

first: Taek Soon

last: Lee

address: 5885 Hollis Street

institute: Joint BioEnergy Institute

summary: Isopentenyl pyrophosphate (IPP) is the universal C5 precursor for isoprenoids, the largest class of natural products and frequent targets for metabolic engineering. In engineered microbial systems, IPP toxicity presents a challenge since its formation is unavoidable in the production of high-value, long-chain terpenes. In this work, we develop an experimental platform to study IPP toxicity in E. coli engineered to produce isoprenol, an IPP-derived C5 alcohol. We first characterize the physiological response to IPP accumulation, demonstrating that elevated levels of IPP are linked to growth inhibition, altered morphology, and reduced cell viability. We show that IPP toxicity selects for pathway “breakage”, using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which results in inhibited nutrient uptake, reduced ATP levels, and an apparent “pause” in metabolism. Finally, we suggest that IPP toxicity is mediated by the formation of an isoprenyl-ATP analog (ApppI). The complementary data presented here represent the most comprehensive assessment of IPP stress to date and suggest potential strategies for the alleviation of IPP and prenyl diphosphate toxicity.

overall_design: To investigate the physiological reason for the metabolic “pause” and subsequent recovery, we used RNAseq to identify differentially expressed genes between engineered strains and the wild-type control at time points corresponding with IPP accumulation and reduction in strain 3Amk. We sampled each strain prior to pathway induction (t=0), at the onset of IPP toxicity in strain 3Amk (t=1), and during growth recovery in strain 3Amk (t=12). By comparing the transcriptome of all 3 strains, we hoped to isolate the impact of IPP accumulation (differentially expressed genes in strain 3Amk only) from the impact of heterologous protein expression and metabolic burden (differentially expressed genes in both 3A and 3Amk relative to WT).

first: Kevin

last: George

first: Mitchell

last: Thompson

first: Joonhoon

last: Kim

first: Taek-Soon


title: Transcriptome analysis of the transcriptional regulator YthA effecting on lactococcus lactis

city: tianjin

first: liu

last: jingui

address: jinnan district yanguanroad tianjin univercity

institute: tianjin univvercity

summary: Our transcriptome data shows that ythA, which is a PspC family transcriptional regulator, obviously affects amino acid metabolism, pyrimidine biosynthesis and nisin immunity.

overall_design: 2 samples, overexpressing ythA strain and the control

first: Hao

last: Wu

first: Jingui

last: Liu

first: Jianjun

last: Qiao


title: Caloric Restriction engages hepatic RNA processing mechanisms in rhesus monkeys

city: Madison

first: Rozalyn

last: Anderson

state: WI

address: 2500 Overlook Terrace

department: Department of Medicine

institute: University of Wisconsin-Madison

summary: Caloric Restriction (CR) extends lifespan and delays the onset of age-related disorders in diverse species. Metabolic regulatory pathways have been implicated in the mechanisms of CR, but the molecular details have not been elucidated. Here we show that CR engages RNA processing of genes associated with a highly integrated reprogramming of hepatic metabolism. We conducted molecular profiling of liver biopsies collected from adult male rhesus monkeys (Macaca mulatta) at baseline and after 2 years on control or CR (30% restricted) diet. Quantitation of over 20,000 molecules from the hepatic transcriptome, proteome, and metabolome, indicated that metabolism and RNA processing are major features of the response to CR. Predictive models identified lipid, branched chain amino acid, and short-chain carbon metabolic pathways, with alternate transcript use for over half of the genes in the CR network. We conclude that RNA-based mechanisms are central to the CR response and integral in metabolic reprograming.

overall_design: The RNA-Seq data set consists of samples derived from liver tissue of 10 animals (5 on control diet, 5 on CR diet). Two biopsy samples were analyzed for each animal: one taken upon enrollment in the CR study, and one taken after two years of study diet.

first: Timothy

last: Rhoads

first: Maggie

middle: S

last: Burhans

first: Vincent

middle: B

last: Chen

first: Paul

middle: D

last: Hutchins

first: Matthew

last: Rush

first: Josef

middle: P

last: Clark

first: Jaime

last: Stark

first: Sean

last: McIlwain

first: Hamid

middle: R

last: Eghbalnia

first: Derek

last: Pavelec

first: Irene

last: Ong

first: John

last: Denu

last: Markley

first: Joshua

last: Coon

first: Ricki

last: Colman


title: Retinal vein occlusion induced by laser treatment in mice

city: Freiburg

first: Gottfried

last: Martin

address: Killianstr. 5

department: Klinik für Augenheilkunde

institute: Universitätsklinikum Freiburg

summary:
Purpose: Several animal models are available for investigating retinal vein occlusion (RVO). Laser-induced RVO has been studied in pigs, rabbits, and rats. Mice have been rarely used despite the huge number of available mutants, ease of handling and cost effectiveness. The aim of this study was to find out if the RVO mouse model is useful for investigating effects of hypoxia.
Methods: C57Bl/6J mice were injected with eosin Y for photo-sensitization. Subsequently, large retinal veins were laser-treated in one eye to induce vascular occlusion. Contralateral control eyes received non-occlusive laser treatment of the retina sparing large vessels. The animals were followed for up to eight days and assessed by fundoscopy, angiography, hypoxyprobe staining, histopathology and gene expression analysis by qPCR and RNA sequencing. Another group of mice was left untreated and studied at a single time point to determine baseline characteristics.
Results: In the RVO group, half of the retinal veins stayed occluded for three days, and one third did so for the whole test period of 8 days. Hypoxia was observed in all RVO eyes for up to 5 days with a maximum around days 2-3. Large retinal edematous areas were present in all laser-treated eyes irrespective of vascular occlusion status. Accordingly, expression of genes associated with inflammation or cell damage was highly up-regulated in both groups as compared to untreated eyes while expression of genes associated with angiogenesis or hypoxia did not change much.
Conclusion: In the laser-induced RVO mouse model, general tissue damage and inflammation responses predominate and obscure specific hypoxia- or angiogenesis-related changes. A non-occlusive control laser treatment is essential to allow for proper data interpretation.

overall_design: Six retinas from 6 mice were combined to one sample. Three replicate retinal tissue samples were obtained in each treatment group (laser treatment of all veins (RVO) or equivalent laser treatment between large vessels (Control)) two days after laser treatment. Three equivalent samples were obtained from untreated mice.


title: Nickel exposure induces persistent mesenchymal phenotype in human lung epithelial cells through epigenetic activation of ZEB1

city: Tuxedo

first: Suresh

last: Cuddapah

state: New York

address: 57 Old Forge Road

department: Environmental Sciecne

institute: New York University Langone Medical center

summary:
Purpose: Environmentally induced diseases, including cancer typically develop long after the exposure has occurred. However, most of the toxicological studies are conducted during active exposure. Therefore, environmental exposure-induced adverse effects that persist after cessation of exposure is poorly understood.
Methods: Immortalized human bronchial epithelial cells (BEAS-2B) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Cellgro) supplemented with 1% Penicillin Streptomycin and 10% Fetal Bovine Serum (FBS, Atlanta Biologicals) at 37 degree C and 5 % CO2. For Ni exposure, various concentrations of NiCl2 (Sigma N6136) was added to the media and the cells were cultured for 6 weeks. Following exposure, the cells were washed and cultured in Ni-free medium for 2 weeks to obtain the Ni-washed-out cells. To obtain homogenous populations of Ni-exposed cells, the cells exposed to 100 mM NiCl2 for 6 weeks were plated in 15 cm plates in Ni-free medium at the rate of 1000, 500, 100 and 50 cells per plate. Nicely separated single colonies were picked and the populations were expanded in Ni-free medium.
Results: Here we report that Ni, an environmentally prevalent, low-mutagenic carcinogen causes epithelial-mesenchymal transition (EMT) that persists even after the exposure has ceased. Ni-induced persistent EMT induction is dependent on the upregulation of ZEB1, an EMT master regulator. We found that Ni exposure resolved the bivalent chromatin environment of ZEB1 gene, thereby keeping it in a permanent ‘on’ state. The continuously expressing ZEB1 downregulated its negative regulators, thus establishing a self-sustaining positive regulatory loop. Given the importance of EMT in a number of diseases including asthma, pulmonary fibrosis, development of premalignancy, carcinogenesis and metastasis, we believe that persistent EMT induction through epigenetic activation of ZEB1 is a major step in Ni-induced human diseases.

overall_design: RNA Seq gene expression comparison done in replicates

first: Lakshmanan

last: Jagannathan

first: Cynthia

last: Jose

first: Vinay

last: Talwar

first: Xiaoru

last: Zhang

first: Chongzhi

last: Zang


title: RNA-Sequencing Analysis of the Effects of UVA Radiation on Keratinocytes

city: Chicago

first: Yu-Ying

last: He

state: IL

address: 5841 S Maryland Ave, AMB N011

institute: University of Chicago

summary: Normal human epidermal keratinocytes were treated with sham or UVA (20J/cm2) radiation in duplicate. 6h after irradiation, samples were purified using Qiagen Rneasy kit, RNA quality assessment was performed using Agilent Bioanalyzer, and library preparation was performed using oligodT selection. RNA-Seq was performed on Illumina HiSeq2000 platform with 50 bp single-end reads. Analysis used the Tuxedo pipeline. Results were validated by qRT-PCR.

overall_design: Transcriptome of sham and UVA-irradiated normal human epidermal keratinocytes

first: Ashley

last: Sample


title: Molecular transitions in early progenitors during human cord blood hematopoiesis

city: Boston

first: Soumya

last: Badrinath

state: MA

address: One Jimmy Fund Way

institute: Dana-Farber Cancer Institute

summary: Hematopoietic stem cells give rise to diverse cell types in the blood system, yet our molecular understanding of the earliest fate decisions that generate this enormous diversity in humans remains incomplete. Here we leverage Drop-seq to individually profile more than 20,000 progenitors cells from human cord blood, without prior enrichment or depletion for individual lineages based on surface markers. Our data reveal a transcriptional 'atlas' of progenitor states in human cord blood, which we leverage to reconstruct differentiation trajectories from HSC to four downstream lineages. And we also demonstrate that Drop-seq data can be utilized to identify new heterogeneous markers of cell state.

overall_design: CD34+ cells were enriched from human cord blood mononuclear cells, and mRNA profiles were generated by paired-end sequencing in Illumina HiSeq, with cDNA being amplified using the Drop-seq protocol, or a modified SMART-Seq2 protocol. Generally, 2-3 replicates were performed per biological sample.

first: Shiwei

last: Zheng


title: The transcription start site and enhancer landscape of the descending colon in inflammatory bowel disease

city: Copenhagen N

first: Morana

last: Vitezic

address: Ole Maaloes Vej 5

department: Department of Biology

country: Denmark

institute: University of Copenhagen

summary: Inflammatory bowel disease (IBD) is a common and chronic gut disorder, with two subtypes: Crohn's disease (CD) and ulcerative colitis (UC), which are challenging to diagnose. The molecular pathology IBD is not well understood, and the underlying gene regulatory regions have not been comprehensively investigated. Relatedly, most IBD-associated SNPs are located in non-coding regions, and may effect gene regulation. Here, we profiled genome-wide promoter and enhancer activity in the descending colon of IBD patients. IBD-induced enhancer and promoters are highly enriched for IBD-associated SNPs, and can predict IBD diagnosis with an accuracy of 85% in an external cohort.

overall_design: This experiment includes a total of 94 patients: 25 patients with active UC(UCa), 17 patients with UC in remission(UCi), 20 patients with active CD(CDa), 3 patients with CD in remission(CDi) and 29 healthy controls (Ctrl).

first: Albin

last: Sandelin


title: H4K20me3 methyltransferase SMYD5 controls heterochromatin and chromosome integrity during embryonic stem cell differentiation [DNA-seq]

city: Detroit

first: Benjamin

last: Kidder

state: MI

address: 4100 John R St

department: Oncology

institute: Wayne State University

summary: Epigenetic regulation of chromatin states is thought to control gene expression programs during lineage specification. However, the roles of repressive histone modifications such as trimethylated histone lysine 20 (H4K20me3) in development and genome stability are largely unknown. Here, we show that depletion of SMYD5, a H4K20me3 methyltransferase, leads to decreased H4K20me3 and H3K9me3 ChIP-Seq levels, and de-repression of endogenous LTR/LINE elements during differentiation. SMYD5 depletion results in chromosomal aberrations and the formation of transformed cells that exhibit decreased H4K20me3 and H3K9me3 levels and an expression signature consistent with multiple human cancers. Moreover, dysregulated gene expression in SMYD5 cancer cells is associated with LTR/ERV elements and decreased H4K20me3. These findings implicate an important role for SMYD5 in maintaining chromosome integrity by regulating heterochromatin and repressing endogenous repetitive DNA elements during differentiation.

overall_design: DNA-Seq of murine shLuc ES cells and shSmyd5 cancer cells.

first: Keji

last: Zhao


title: Transcriptional profiling reveals gene programs that are differentially regulated in cardiac fibroblasts during exercise and disease

city: Rochester

first: Eric

last: Small

address: 601 Elmwood Avenue, Box CVRI

institute: University of Rochester

summary:
Purpose: Cardiac fibroblasts (CFs) contribute to pathological remodeling by proliferating and secreting extracellular matrix.  In contrast, physiological remodeling is an adaptive response to excessive demands of exercise that results in increased heart size without accompany fibrosis or deterioration of cardiac function.  We hypothesize that these divergent outcomes are partially a function of cell-autonomous and paracrine cardioprotection afforded by CFs specifically during physiological remodeling.  Our objective is to define the gene expression program (GEP) of CFs isolated from mouse models of physiological (swim) and pathological (thoracic aortic constriction (TAC) or myocardial infarction (MI)) remodeling to identify novel mechanisms underlying the development of pathological cardiac fibrosis and the cardioprotective benefits of exercise.
Methods: Cardiomyocytes (CMs) were isolated by gravity sedimentation and CFs were isolated by differential plating for 2 hours from single-cell suspensions of 10-14 week old male C57BL/6 hearts subjected to the following 7 conditions: sedentary CFs (n=3), sham surgery CFs (n=2), 3 day TAC CFs (n=3), 10 day TAC CFs (n=3), 4 day swim CFs (n=3), 10 day swim CFs (n=3), and 5 day MI CFs (n=2).  CMs were isolated from sedentary 10-14 week old male C57BL/6 hearts (n=3).  Total RNA was isolated using the Qiagen RNeasy Plus Kit and genomic DNA removed using Qiagen gDNA columns. RNA quality was assessed with the Agilent Bioanalyzer and only samples with RIN >8.5 were used for sequencing. Library construction was performed using the TruSeq Stranded mRNA Sample Preparation Kit from 100ng poly-A mRNA.  An Illumina HiSeq 2500 was used to generate 20-25 million single end reads of 100nt for each sample.  The sequence reads that passed quality filters (Trimmomatic-0.32) were analyzed at the transcript isoform level using STAR_2.4.2a for SHRiMP_2_2_3 mapping to mouse genome assembly GRCm38.p4 version mm10 and processed with Cufflinks-2.0.2.
Results: We determined that CFs display distinct GEPs that are inversely regulated in pathological and physiological remodeling.  Spearman correlation and Principal Component Analysis (PCA) of genes with FPKM >1 and q >0.05 revealed consistency between biological replicates and highlighted active changes in GEPs during physiological and pathological remodeling both of which diverged from control samples in principal component (PC) 1 and PC2.  CFs isolated from mice 5 days after initiation of MI also diverged from controls, TAC, and swim samples, demonstrating a different fibroblast GEP in ischemic and pressure overload induced remodeling.  This analysis also revealed that our control models (sham surgery and sedentary) were indistinguishable from each other, therefore we typically treated sham and sedentary mice as a single control group in further studies.  These initial results reveal that CFs differentially respond to pathological and physiological cues in as few as 3 days by enacting dynamic transcriptional changes in vivo.  Consistent with previous reports of myofibroblast activation, Ingenuity Pathway Analysis (IPA) revealed that the most significantly enriched GO terms in pathological fibroblasts included stimulation of Rho-dependent contractile pathways and integrin-linked kinase (ILK) signaling, the latter of which was downregulated in a comparison between 10-day swim vs. control CFs.  IPA transcription factor analysis comparing 10-day swim and 10-day TAC CFs further identified upregulation of detoxification and inflammatory transcription factors and downregulation of the serum response factor (SRF) after exercise.  Furthermore, independent motif analysis using HOMER 2.0 identified the CArG box (SRF binding site) as the most enriched transcription factor binding site within 5kb of genes that are upregulated in 10-day TAC vs sham CFs.
Conclusions: We have defined the GEPs of CFs that underlie physiological and pathological cardiac remodeling.  Not only is the GEP of pathological CFs enriched in Rho-dependent pathways and SRF target genes, but the GEP of physiological CFs is enriched in detoxification and inflammatory target genes.  We also demonstrate that the CFs change their transcriptional signature consistently and rapidly.  Within 3-4 days of initiating extracellular stimuli, distinct profiles are observed in CFs isolated from swim-trained, pressure overload, or ischemic models of cardiac remodeling.

overall_design: Poly-A RNA from cardiac fibroblasts and cardiomyocytes across 8 conditions (sedentary CF, 3 day sham CF, 3 day TAC CF, 10 day TAC CF, 5 day MI, 4 day swim CF, 10 day swim CF, sedentary CM) were submitted for deep sequencing using an Illumina HiSeq 2500.

first: Janet

last: Lighthouse


title: Define roles of JMJD1B in mediating histone demethylation and gene expression

city: Duarte

first: Juan

last: Du

address: 1500 E. Duarte Rd

institute: Beckman Research Institute, City of Hope

summary: The arginine or lysine methylation status of histones dynamically changes during many essential cellular processes, particularly during embryonic and hematopoietic stem cell development. The enzymes demethylate methyllysine residues have been well defined, but the enzymes demethylate the methylarginine residues during different cellular processes are unknown. In current study, we demonstrate that JMJD1B is a lysine demethylase for H3K9me2 and an arginine demethylase for H4R3me2s. To reveal the biological significance of JMJD1B as an arginine demethylase, we isolate hematopoietic stem/progenitor cells (HSPC) from wild-type and JMJD1B knockout (JKO) bone marrow. We then conduct ChIP-seq on H4R3me2s and H3K9me2 histone markers and perform RNA-seq to determine the global gene expression profiles. We have observed global demethylation of H4R3me2s at the gene body but not the intergenic regions in hematopoietic stem/progenitor cells. H4R3me2s demethylation at the gene body region is directly correlated with gene expression in these cells. Furthermore, knockout of JMJD1B causes defects in removing the H4R3me2s epigenetic marker, leading to down-regulation of genes important for blood cells differentiation and development. Altogether, our current study demonstrates that arginine demethylases exist in cellular systems and that JMJD1B demethylates H4R3me2s for proper epigenetic programming during development.

overall_design: For each genetic background, a pool of HSPCs and BMCs from four biological replicates are collected and analyzed by ChIP-seq. HSPCs from two biological replicates are analyzed by RNA-seq. For ChIP-seq, the input and/or ChIP histone H4 is used as control.

first: Li

first: Mian

last: Zhou

first: Ali

last: Shafat

first: Xiwei

last: Wu

first: Binghui

last: Shen

first: Yang

last: Lu


title: Circulating miRNAs. isomiRs and small RNA clusters in human plasma and breast milk

first: Lorena

last: Pantano

address: 655 Huntington Ave

department: Biostatistics

institute: Harvard Chan School of Public Health

type: Non-coding RNA profiling by high throughput sequencing

summary: Circulating small RNAs. including miRNAs but also isomiRs and other RNA species. have the potential to be used as non-invasive biomarkers for communicable and non-communicable diseases. This study aims to characterize miRNAs. isomiRs and small RNA clusters in biofluids. to compare their profiles. and to help in the establishment of standard guidelines. For this purpose. RNA from plasma and breast milk samples from 15 healthy postpartum mothers was extracted. Small RNA libraries were prepared with the NEBNext® small RNA library preparation kit and sequenced in an Illumina HiSeq2000 platform. After an initial quality control. miRNAs. isomiRs and clusters of small RNAs were annotated using seqBuster/seqCluster framework. The average amount of extracted RNA was 81 ng/mL [standard deviation (SD): 41] and 3985 ng/mL (SD: 3767) for plasma and breast milk. respectively. Mean number of good quality reads was 4.04 million (M) (40.01% of the reads) in plasma and 12.5M (89.6%) in breast milk. One thousand one hundred eighty two miRNAs. 74.317 isomiRs and 1.053 small RNA clusters that included piwi-interfering RNAs (piRNAs). tRNAs. small nucleolar RNAs (snoRNA) and small nuclear RNAs (snRNAs) were detected. Samples grouped by biofluid. with 308 miRNAs. 4.737 isomiRs and 778 small RNA clusters differentially detected. In summary. plasma and milk showed a completely different small RNA profile. In both. miRNAs. piRNAs. tRNAs. snRNAs. and snoRNAs were identified. confirming the presence of non-miRNA species in biofluids

overall_design: Peripheral blood was collected at 0-48 h post-partum for 15 participants. only 5 samples of plasma pass QC; and breast milk was collected at 48-72 h post-partum for 10 of the participants

first: Mercedes

last: Rubio

first: Mariona

last: Bustamante

first: Carles

last: Hernandez-Ferrer

first: Yaris

last: Sarria

first: Maria

last: Piqué-Borras

first: Kilian

last: Vellve

first: Silvia

last: Agramunt

first: Ramon

last: Carreras

first: Xavier

last: Estivill

last: Gonzalez

first: Alfredo

last: Mayor


title: Jurkat cell exosomes bound by  CD47, CD63 and MHC1 harbor different RNA content

status: Public on Mar 13 2018

city: Bethesda

first: Sukhbir

last: Kaur

address: 10 Center Drive

department: NCI

institute: NIH

summary: Extracellular vesicles (EVs) mediate cell-cell communication including the intercellular transfer of RNAs, which alter gene expression in target cells. However, heterogeneity in the size, density, and composition of EVs has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC-1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing the properties of Jurkat T cell EVs captured using CD47, CD63, and MHC-1 antibodies. These EV subsets have similar sizes but divergent RNA contents.

overall_design: Jurkat cell exosomes where bound by CD47, CD63 and MHC1 antibodies and compared to their un-bound exosomes using a total of 14 samples: triplicates for CD47 bound and unbound and duplicates for the rest.

first: Abdel

last: Elkahloun

first: Timothy

last: Myers

first: Lynn

last: Young


title: HDAC1/2 are required for microglia identity during development, homeostasis and neurodegeneration in a context-dependent manner [ChIP-seq constitutive knockout]

first: Ori

last: Staszewski

address: Breisacher Str. 64

department: Institute of Neuropathology

institute: University Medical Center Freiburg

summary: Microglia as tissue macrophages of the central nervous system (CNS) provide immunological defense and contribute to the establishment and maintenance of CNS homeostasis. Several transcription factors have been described that regulate microglia development and its steady state form, however little is known about the epigenetic signals that control microglia function in vivo. Here, we employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases (HDACs), Hdac1 and 2. Prenatal ablation of Hdac1 and 2 from microglia drastically impaired microglial development with reduced cell number, altered morphology and induction of apoptosis. Comparative transcriptomic profiling revealed an essential role of Hdac1/2 in the regulation of microglia survival and expansion. Mechanistically, hyperacetylation at H3K9 and H3K27 was found at the promoters of pro-apoptotic and cell cycle genes in the absence of Hdac1/2. In contrast, Hdac1/2 were not required in adult microglia during homeostasis. In a mouse model of Alzheimer’s disease, suppression of Hdac1/2 in microglia, but not in neuroectodermal cells, remarkably displayed substantial decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a previously unknown role of epigenetic factors that differentially affect microglia development, homeostasis and disease that could potentially be utilized therapeutically.

overall_design: HDAC1/2-2lox-Cx3cr1-Cre microglia were sorted and ChIP was performed for histone marks H3K9ac and H3K27ac. Samples were compared to wildtpye controls using 2 or 3 replicates.

first: Moumita

last: Datta

first: Elena

last: Raschi

first: Maximilian

last: Frosch

first: Nora

last: Hagemeyer

first: Tuan

last: Tay

first: Thomas

last: Blank

first: Melanie

last: Meyer-Luehmann

first: Marco

last: Prinz


title: High throughput screen identifies novel small molecules that derepress genes repressed by DNA methylation

status: Public on Mar 12 2018

city: Beijing

first: Zhuqiang

last: Zhang

address: 15 Datun Road, Chaoyang District.

institute: Institute of Biophysics, CAS

summary: Genes repressed by DNA methylation can be derepressed by various compounds targeting DNA methyltransferases, histone deacetylases, etc. Here, we report a high throughput screen against a 308,251-member chemical library aiming to identify novel small molecules that derepress an EGFP reporter gene silenced by DNA methylation. RNA-seq was used to measure mRNA abundance in control and compound-treated cells.

overall_design: RNA-seq was used to measure mRNA abundance in DMSO-treated cell, 5aza-dC treated cells, RG108-treated cells and 7 top hits novel compunds treated cells. MeDIP-seq was also done in untreated control cells.

first: Xiang

last: Li

first: Erchang

last: Shang

first: Qiang

last: Dong

first: Yingfeng

first: Jing

first: Shaohua

last: Xu

first: Zuodong

last: Zhao

first: Wei

last: Shao

first: Hua

last: Huang

first: Yong

last: Zheng

first: Hailin

last: Wang

first: Xiaoguang

last: Lei

first: Bing

last: Zhu


title: Detection of different expressed genes in newborn piglet liver using RNA-seq to analyze negative effects caused by dietary tryptophan

city: Changsha

first: Kang

state: Hunan

address: 644 Yuanda 2 Road

institute: Institute of Subtropical Agriculture, Chinese Academy of Sciences

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of the expression of genes. The goals of this study are to reveal the mechanism of side effect of excess diet tryptophan in newborn piglets through optimal high-throughput data analysis.
Methods: Liver mRNA profiles of newborn piglets feeded by different tryptophan mode were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the genes level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks and Then, differential expression genes analysis between these two groups (three biological replicates per condition) was performed using Cuffdiff. The P values were adjusted using the Benjamini & Hochberg method. qRT–PCR validation was performed using TaqMan and SYBR Green assays
Results: Using an optimized data analysis workflow, we mapped about 30 to 40 million sequence reads per sample to the pig genome (build susScr3) and identified 11,314 transcripts in the liver of newborn piglets with TopHat workflow. RNA-seq data confirmed 88 different expressed genes between the High-Low group and the 2×Trp group, and 10 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR.
Conclusions: Our study represents the first detailed analysis of liver transcriptomes of newborn piglets from the groups that sow feed by High-Low and the 2×Trp methods, with biologic replicates, generated by RNA-seq technology. Functional analyses of DEGs in livers revealed that certain genes associated with cellular stress and exogenous stimulus response were strongly associated with the dysfunction of liver metabolism in newborn piglets.

overall_design: Liver mRNA profiles of newborn piglets were generated by deep sequencing, in triplicate, using Illumina 2500.

first: Yulong

last: Yin


title: Impact of B16F0 exosomes on the transcriptome of CTLL2 cytotoxic T cells

city: Morgantown

first: David

middle: John

last: Klinke

state: WV

department: Chemical Engineering

institute: West Virginia University

summary: While recent clinical studies demonstrate the promise of cancer immunotherapy, a barrier for broadening the clinical benefit is identifying how tumors locally suppress cytotoxic immunity. As an emerging mode of intercellular communication, exosomes secreted by malignant cells can deliver a complex payload of coding and non-coding RNA to cells within the tumor microenvironment. Here, we quantified the RNA payload within tumor-derived exosomes and the resulting dynamic transcriptomic response to cytotoxic T cells upon exosome delivery to better understand how tumor-derived exosomes can alter immune cell function. Exosomes derived from B16F0 melanoma cells were enriched for a subset of coding and non-coding RNAs that did not reflect the abundance in the parental cell. Upon exosome delivery, RNAseq revealed the dynamic changes in the transcriptome of CTLL2 cytotoxic T cells. In analyzing transiently co-expressed gene clusters, pathway enrichment suggested that the B16F0 exosomal payload altered mitochondrial respiration, which was confirmed independently, and upregulated genes associated with the Notch signaling pathway. Interestingly, exosomal miRNA appeared to have no systematic effect on downregulating target mRNA levels.

overall_design: CTLL2 cells were grown in complete media for 24 hrs, and then stimulated with fresh B16F0 exosomes resuspended in PBS, to a final exosome concentration of 0.2 mg/ml. The transcriptome of untreated CTLL2 cells was assayed at 0, 0.5, 2, 4, and 8 hours after cells were placed in fresh media. There are 4 biological replicates at the 0 hour time point and 3 biological replicates at the 0.5, 2, 4, and 8 hour time points. The transcriptome of CTLL2 cells treated with B16F0 exosomes was assayed at 0.5, 2, 4, and 8 hours after addition of fresh media containing B16F0 exosomes. There were 3 biological replicates performed at each time point.


title: Phenotypic and microRNA characterisation of the hidden CD24+ cell population in MCF-7 breast cancer 3D spheroid culture

city: Cheras

first: norlaily

last: mohd ali

state: Selangor

address: Universiti Tunku Abdul Rahman, Jln Sg Long

department: Faculty of Medicine and Health Sciences

country: Malaysia

institute: Universiti Tunku Abdul Rahman

summary:
Introduction: In vitro 3-dimension spheroid culture has been widely used as model to enrich CD44+CD24dim/- cancer stem cells with high ALDH1 activity. However, present of the CD24+ population in the 3D spheroid also requests further attention as this side-population of cells may response differently to drug treatment comparing to the cancer stem cells.
Methods: In this study, CD24+ population from the MCF-7 spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion/migration test, and microRNA expression profiling. MCF-7 CD24+ cells sorted from primary spheroid reform 3D spheroid after longer period of time associated with dim expression of surface SOX-2, CD44, CD49f and Nanog comparing to the primary spheroid.
Results: Remarkably, the MCF-7 CD24+ cells was found with high expression of ALDH1 protein, which subsequently contributed to higher resistant against doxorubicin and cisplatin. MicroRNA profiling has shown that absent of cancer stem cell phenotypes were contributed by the downregulation of major cancer stem cells related pathways including Hedgehog, Wnt and MAPK signalling pathways. Besides, resistance of CD24+ cells was correlated to dyregulation of cell death pathway.

overall_design: miRNA profiles of MCF-7 CD24+ cells spheroids and monolayer cultures were generated in triplicates by deep sequencing using TruSeq small RNA illumina

first: Lily

last: Boo

first: Swee

middle: K

last: Yeap

first: Norlaily

last: Ali

first: Wan

middle: Y

last: Ho

first: Huynh

last: Ky

first: Kok

last: Chan

first: Wai

middle: F

first: Dilan

middle: A

last: Satharasinghe

first: Woan

middle: C

last: Liew

first: Sheau

last: Tan

first: Soon

last: Cheong

first: Han

last: Ong


title: Puf3 participates in ribosomal biogenesis in malaria parasites

status: Public on Mar 11 2018

city: State College

first: Liwang

last: Cui

state: PA

address: 553 ASI bldg

institute: Penn State

summary: Recent studies of the Puf family of RNA-binding proteins revealed that besides their traditional roles in translational regulation of mRNAs, some Puf proteins are also involved in ribosome biogenesis by binding rRNA. In this study, we report the role of a Puf-like protein in Plasmodium falciparum (name as PfPuf3) and its ortholog PyPuf3 in Plasmodium yoelii in ribosome biogenesis. Secondary structure prediction suggested that the RNA-binding domain of Puf3 consists of 11 pumilio repeats, similar to human Puf-A/yeast Puf6, which involved in ribosome biogenesis. Neither pfpuf3 nor pypuf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle (IDC). A time-course study of PfPuf3 protein indicated that PfPuf3 was expressed during the entire IDC, with peak expression in early trophozoites. Cellular fractionation of PfPuf3 revealed that it is preferentially partitioned to the nuclear than the cytoplasmic fractions, which is consistent with a nuclear localization of PfPuf3::GFP and PyPuf3::GFP as seen by immunofluorescence. Further, we found PfPuf3 co-localized with a well-known nucleolus maker, PfNop1, demonstrating that PfPuf3 is a nucleolar protein. This localization is in contrast to the cytoplasmic localization of PfPuf1 and PfPuf2, but matches the localization of human Puf-A and yeast Puf6. Affinity purification of an N-terminal PTP-tagged variant of PfPuf3 revealed an association with 32 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and ITS2. Taken together, these results demonstrate a nucleolar localization of PfPuf3 and suggest an essential function of PfPuf3 in ribosomal biogenesis.

overall_design: The transgenic parasite line PfPuf3-PTP was employed for RIP and RNA-seq analysis in order to indentify Puf3 targets.To control for nonspecific RNA binding, the same experiments were perfomred with wild-type 3D7 parasite. Reads comparsions of ribosomal RNAs before and after purification were calculated.

first: Xiaoying

last: Liang

first: Kevin

last: Hart

first: Gang

last: Dong

first: Faiza

last: Siddiqui

first: Aswathy

last: Sebastian

first: Xiaolian

last: Li

first: Istvan

last: Albert

first: Jun

last: Miao

first: Scott

middle: E

last: Lindner


title: PCGF5 is required for neural differentiation of embryonic stem cells [RNA-seq]

status: Public on Mar 10 2018

city: Guangzhou

first: Jiajian

last: Zhou

state: Guangdong

address: No. 2 Lujing Road, Yuexiu District,

department: Dermatology Hospital

institute: Southern Medical University

summary: Polycomb-group proteins are key regulators of the transcriptional programs that maintain stem cell identity and dictate lineage specification. Polycomb repressor complex 1 (PRC1) contains the E3 ligases RING1A/B, which monoubiquitinate lysine 119 at histone H2A (H2AK119ub1) to regulate gene expression. PRC1 has been sub-classified into six major complexes based on the presence of a PCGF subunit. Here, we find that Pcgf5, one of six PCGF paralogs, is an important requirement in the differentiation of mouse embryonic stem cells (mESCs) towards a neural cell fate. Although PCGF5 is not required for mESC self-renewal, its loss blocks mESC neural differentiation by activating the SMAD2/TGF-β signaling pathway. Inhibition of SMAD2/TGF-β signaling or rescue by overexpression of Pcgf5 can restore the capability of mESCs to differentiate towards a neural cell fate. PCGF5 works by stimulating RING1B-dependent H2AK119ub1 both in vitro and in vivo, leading to the suppression of TGF-β signaling genes. PCGF5 loss-of-function prevents the reduction of H2AK119ub1 and H3K27me3 around neural specific genes and keeps them repressed. Our results showed that PCGF5 might function as both a repressor for SMAD2/TGF-β signaling pathway and a facilitator for neural differentiation. Together, our findings reveal a critical context-specific function for PCGF5 in directing PRC1 to control cell fate.

overall_design: Both wild type and Pcgf5-/- mESCs were induced to differentiate in N2B27 medium for 6 days. Then the cells were harvested at day 0 and day 6 after neural differentiation with 2 biological replicates. Total RNAs were collected for either RT-qPCR or RNA sequencing.

first: Mingze

last: Yao

first: Xueke

first: Shixin

last: Gong

first: Gongcheng

last: Hu

first: Jiao

first: Kaimeng

last: Huang

first: Ping

last: Lai

first: Guang

last: Shi

first: Andrew

last: Hutchins

first: Hao

last: Sun

first: Huating

last: Wang

first: Hongjie


title: KAP1 regulates Treg function and proliferation.

city: Chiba

first: Shigeru

last: Tanaka

address: 1-8-1 Inohana

department: Allergy and Clinical Immunology

institute: Chiba University

summary: KAP1 is a partner of Foxp3 in Tregs. Since Treg specific KAP1 deficient mice develop autoimmune disease, KAP1 is an important factor. To address the mechanisms of KAP1 regulation in Treg function, we performed RNA-seq analysis.

overall_design: mRNA of KAP1 sufficient and deficient Tregs were sequenced using Hiseq2500.

first: Steven

last: Ziegler


title: H3K9Me3 marks in KAP1 deficient regulatory T cells

summary: We aim to determine the role of KAP1 in regulatory T cells. Sicne KAP1 recruits H3K9 specific methyltransferase SETDB1, we focused on H3K9me3. Although H3K9me3 marks aroung transcription start site were dramatically reduced, this change did not correlated with the change of transcription levels between KAP1 sufficient and deficient Tregs determined by RNA-seq. Our data suggest the transcritpion regulation by KAP1 in regulatory T cells is not dependent on H3K9me3 marks.

overall_design: KAP1 sufficient and deficient Tregs are sorted and the chromatin was immunoprecipitated with anti-H3K9Me3 antibody. The precipitated chromatin was sequenced by Hiseq2500.


title: PCGF5 is required for neural differentiation of embryonic stem cells [ChIP-seq]

overall_design: H2AK119ub1 and H3K27me3 ChIP-seq with a drosophila chromatin spike-in were performed in wild type, PCGF5-depleted mESCs and the differentiated NPCs at day 6 after neural differentiation of mESCs, respectively. PCGF5 ChIP-seq was performed in NPCs at day 6 after neural differentiation of mESCs.


title: Constitutive and Tamoxifen inducible knockout of HDAC1 and HDAC2 in Microglia

first: Ori

last: Staszewski

summary: Hdac1 and 2 are important regulators of developmental processes. In this study we created microglia specific compound Hdac1 and Hdac2 knock out mice. Pre-natal ablation of both Hdac1 and 2 from microglia leads to reduced cell number and altered cell morphology. To investigate how Hdac1 and 2 knock out in microglia alters cellular gene expression profile we carried out RNA-seq analysis at different time points.

overall_design: We used FACS sorted microglia cells from control and Hdac1/2fl/flCx3cr1Cre (constituitive knockout) or Hdac1/2fl/flCx3cr1CreERT2 (inducible) mice at different time points viz. Embryonic day 16 (E16 - inducible knockout only), Post natal day 0 (P0), 2 and 6 weeks after birth

first: Moumita

last: Datta

first: Marco

last: Prinz


title: Visualizing germinal center B cell dynamics at single cell transcriptome resolution

city: Cincinnati

first: Harinder

last: Singh

state: OH

address: 333 Burnet Avenue

department: Immunobiology

institute: CCHMC

summary: During a germinal center (GC) response, B cells diversify their immunoglobulin (Ig) genes by somatic hyper-mutation (SHM) and undergo clonal expansion and positive selection thereby enabling the generation of higher affinity antibodies. We have analyzed the genomic states underlying GC B cell dynamics by single cell RNA-Seq. Profiling of antigen specific GC B cells during the peak of the response, revealed four distinctive genomic states characterized by antigen presentation, apoptotic, mitochondrial and mitotic gene expression modules. Intersection of genomic states and Ig heavy-chain (Igh) class-switch trajectory suggested that mitochondrial machinery is utilized to support class-switch recombination (CSR). Furthermore, by analyzing the transcriptomes of B cells with varying affinity BCR sequences that assembled from single-cell RNA-seq data through a novel algorithm, we show that high affinity GC B cells manifest enhanced mitotic and BCR signaling transduction, but compromised antigen processing and presentation gene expression modules. Thus, we are developing a comprehensive framework of the genomic states and molecular pathways underlying GC B cell dynamics.

overall_design: C56BL/6J (Jax 664) mice were immunized intraperitoneally with 100 μg NP(23)-KLH (Biosearch Technologies) mixed with 50% (v/v) Alum (Thermo Scientific) and 1 μg LPS (Sigma). Splenocyte suspensions were prepared in MACS buffer (pH 7.4 PBS plus 1% FBS and 5 mM EDTA) on day 13 post immunization and blocked with 25 μg/ml 2.4G2 (BD) for 15 min on ice. Then cells were labeled with 2 μg/ml biotin anti-CD3, 1 μg/ml biotin anti-CD4, 1 μg/ml biotin anti-CD8, 2.5 μg/ml biotin anti-CD11C and 2.5 μg/ml biotin anti-IgD for 25 min at 4°C. After washing four times, cells were further labeled with anti-biotin beads (Miltenyi Biotec) for 20 min at 4°C. The magnetic columns were used to deplete non-GC B cells according to standard protocols (Miltenyi Biotec). The effluent cells were further labeled with NP-PE, B220, Fas and GL7 antibodies (Supplementary Table 1) for 30 min at 4 °C. GC B cells were sorted as 7AAD–B220+Fas+GL7hi with FACSAria II (BD) with 70 μm nozzle. Single GC B cells were prepared using the C1TM Single-Cell Auto Prep System (Fluidigm, San Fransisco, CA), according to the manufacturer’s instructions. 182 scRNA Seq libraries were sequenced per HiSeq 2500 gel with 75bp paired-end sequencing.

first: H

last: Xu

first: A

last: Khan


title: ATAC-seq of sorted lung cDCs (conventional dendritic cells) from cell-specific MyD88 KO at 0h and 6h in vivo sensitization with OVA/standard flagellin

city: RTP

first: Seddon

last: Thomas

state: North Carolina

address: 111 T. W. Alexander Dr.,

institute: NIEHS

summary: Allergic asthma is a chronic disease of the airways characterized by eosinophilic and neutrophilic inflammation. MYD88, the adaptor molecule for TLR and IL-1 family member signaling, is required for allergic sensitization through the airway in animal models of allergic asthma. We generated conditionally mutant mice separately lacking Myd88 in airway epithelial cells (ECs) or dendritic cells (DCs) to define the contribution of Myd88 expression in each of these cell types. To examine epigenetic crosstalk from ECs to conventional (c)DCs in vivo, we examined ATAC-seq data from lung cDCs sorted at baseline or following 6h in vivo lung allergic sensitization through the airways from WT MyD88 fx/fx, SPC cre+ MyD88 fx/fx (EC-MYD88 KO), CD11c cre+ MyD88 fx/fx (DC-MYD88 KO), and full MyD88 KO mice.  We observed specific epigenetic changes in chromatin conformation in cDCs that were cell-intrinsic as well as resulting from in vivo crosstalk from ECs. We also observed transcriptional changes in cDCs by Affymetrix Whole Transcriptome Array (linked data set) as well as changes in immune-specific whole lung RNA, sorted EC RNA, sorted alveolar macrophage RNA, and sorted cDC RNA changes by Nanostring nCounter Immunology Codeset Analysis (additional linked files).

overall_design: Lung cDCs from all genotypes were flow cytometry sorted at baseline or following 6h of in vivo allergic sensitization with 100 micrograms ovalbumin (OVA)/ 1250 ng standard flagellin (stFLA). Samples from all 4 genotypes (WT MyD88 fx, SPC cre+ MyD88 fx, CD11c cre+ MyD88 fx, MyD88 KO) x 2 timepoints (0h baseline, 6 h OVA/stFLA) = 8 conditions. 8 conditions x 2 replicates per genotype-timepoint = 16 total samples. Analysis of baseline (0h) timepoint is a control for induced chromatin conformation changes as compared to those at baseline.  WT MyD88 fx/fx mice are a positive control for all potential chromatin-induced at baseline and following allergen sensitization, while MyD88 KO is a negative control for epigenetic changes that are not dependent on MYD88 expression in any cell-type or tissue. SPC cre+ MyD88 fx/fx mice test for epigenetic changes that require EC-dependent MYD88 signaling, whereas CD11c cre+ MyD88 fx/fx mice test for epigenetic changes that require CD11c-expressing cell-dependent MYD88 expression. ATAC-seq analysis was conducted as described.

first: Donald

middle: N

last: Cook


title: Synthetic lethality between ARID1A mutation and HDAC2 inhibition in ovarian cancer

status: Public on Mar 09 2018

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: ARID1A, encoding a subunit of the SWI/SNF chromatin remodeling complex, is the most frequently mutated epigenetic regulators in human cancers.  ARID1A is mutated in over 50% ovarian clear cell carcinoma, a disease currently has no effective therapy.  Here we show that ARID1A-mutated ovarian cancer cells are selectively sensitive to inhibition of HDAC2 activity. HDAC2 interacts with EZH2 in an ARID1A status dependent manner. HDAC2 knockdown inhibits the growth of ARID1A inactivated by not proficient ovarian cancer cells. HDAC2 functions as a co-repressor of EZH2 to suppress the expression of EZH2/ARID1A target tumor suppressor genes such as PIK3IP1, an inhibitor of PI3K/AKT signaling, to inhibit proliferation and promote apoptosis.  Indeed, a FDA-approved pan-HDAC inhibitor suberoylannilide hydroxamine (SAHA) significantly suppressed the growth and reduced the ascites of the ARID1A-inactivated ovarian cancers in both orthotopic and genetic mouse models.   This correlated with a significant improvement of survival of mice bearing ARID1A-mutated ovarian cancers.

overall_design: HDAC2 wild type and shRNA knockdown samples assayed by RNA-seq

first: Rugang

last: Zhang

first: Takeshi

last: Fukumoto


title: scRNA-seq 8- to 16-cell stage bovine embryo

city: Munich

first: Ilaria

last: Lavagi

address: Feodor Lynen Strasse 25

institute: Ludwid Maximilian University

summary: we analysed the transcriptome of bovine embryo during the major Embryonic Gene Activation (EGA) in cows. We did it at a single cell level to investigate if the cell are heterogeneous and to check if early specification events take place already at this stage.

overall_design:
In vitro fertilization of bovine embryos. Single cell RNA sequencing at 8- and 16-cell stages. SCRB-seq library

Please note that
[1] sample titles represent embryo, blastomere and plate number. For example,  E14.1_p3 means exactly embryo 14, blastomere 1, plate 3.
[2] more than one cell has origin from the same embryo. For example, E2-Day2 (in the source embryo) means Embryo 2 collected at Day 2.

first: Stefan

last: Krebs

first: Kilian

last: Simmet

first: Andrea

last: Beck

first: Valeri

last: Zakhartchenko

first: Eckhard

last: Wolf

first: Helmut

last: Blum


title: MYD88-dependent dendritic and epithelial cell crosstalk in the lung orchestrates immune responses to inhaled allergens

type:
Expression profiling by array
Other

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

overall_design: Refer to individual Series


title: Enhancement of Arterial Specification in Human Pluripotent Stem Cell Cultures Promotes Definitive Hematoendothelial Program with Broad Myelolymphoid Potential

city: Madison

first: Oleg

last: Moskvin

address: 1220 Capitol Court

institute: University of Wisconsin-Madison

summary: Identification of the regulators that lead to arterial specification with definitive hematopoietic potential should help to design strategies to recapitulate HSC development from human pluripotent stem cells (hPSCs). Here, using ETS1 conditional H1 hESC line, we found that ETS1 induction at the mesodermal stage of differentiation dramatically enhances the arterial specification in hPSC cultures and formation of DLL4+CXCR4+/- arterial HE with lymphoid potential and the capacity to produce red blood cells with high expression of BCL11a and b-globin. ETS1 effect was mediated through upregulation of NOTCH signaling. Together, these findings demonstrated that promotion of arterial specification in cultures could aid in generation of definitive hematopoiesis from hPSCs.

overall_design: We engineered H1 human embryonic stem cells (hESC) carrying doxycycline (DOX)-inducible ETS1-P2A-EGFP transgene (iETS1-hESCs) and differentiated them to endothelial and hematopoietic cells in chemically defined conditions

first: Igor

last: Slukvin

first: Mi

last: Park

first: Scott

last: Swanson


title: Efficient Mammary Gland Involution Requires c-Jun N-terminal kinase

city: Worcester

first: Dr Yvonne

last: Edwards

address: 373 Plantation Street

department: Program in Molecular Medicine

institute: University of Massachusetts Medical School

summary: Involution returns the lactating mammary gland to a quiescent state after weaning. The mechanism of involution involves collapse of the mammary epithelial cell compartment. To test whether the cJUN NH2-terminal kinase (JNK) signal transduction pathway contributes to involution, we established mice with JNK deficiency in the mammary epithelium. We found that JNK is required for efficient involution. JNK deficiency did not alter the STAT3/5 or SMAD2/3 signaling pathways that have been previously implicated in this process. Nevertheless, JNK promotes the expression of genes that drive involution, including matrix metalloproteases, cathepsins, and BH3-only proteins. These data demonstrate that JNK has a key role in mammary gland involution post lactation.

overall_design: WAP-Cre and Jnk1f/f Jnk2f/f WAP-Cre mice were bred for a single pregnancy and litters were normalized to 6-8 pups. The pups were allowed to nurse for 9 days before forced weaning. At that point, some mice were euthanized and their mammary glands were harvested to isolate RNA (0 days). Other mice were kept for 3 days before euthanasia and mammary gland harvest (3d). In this way, gene expression differences could be determined between JNK-null and JNK-wildtype mammary glands before and during involution.

first: Girnius

last: Nomeda

first: Davis

last: Roger J


title: RNAseq of quiescent (Q) and stress induced premature senescent (SIPS) fibroblasts treated with plant extract (1201) from Solidago vigaurea subspecies alpestris

city: Vienna

first: Markus

last: Schosserer

address: Waehringer Strasse 10

department: Center for Pathobiochemistry and Genetics

country: Austria

institute: Medical University of Vienna

summary: Quiescent (Q) and stress induced premature senescent (SIPS) fibroblasts were treated for the duration of 4 days with growth medium supplemented with a plant extract (1201) from Solidago vigaurea subspecies alpestris. Rna was isolated with Trizol and sent to GATC for next generation sequencing with Ilumina technology. The plant extract proofed to block the negative effects of senescence in human skin fibroblasts in various experiments by delaying the acquisition of a senescent phenotype/favouring a papillary-like phenotype and attenuating the senescence associated secretory phenotype. The RNAseq was performed to understand the underlying molecular mechanism of the observed effects. SIPS was induced by chronic oxidative stress treatment (9 days with 1 h 100 µM H2O2 per day).

overall_design: 12 samples in total. Quiescent cells (Q), quiescent cells treated with 1201 for 4 days (Q1201), stress induced premature senescent cells (SIPS) and stress induced premature senescent cells treated with 1201 for 4 days (SIPS1201), all conditions in triplicates. Quiescent cells are considered the appropiate control for senescent cells.

first: Ingo

last: Laemmermann


title: Genome-wide profiling of mature piRNAs in human Neuroblastoma (NB) cell lines

status: Public on Mar 20 2018

city: Rourkela

first: Dr. Bibekanand

last: Mallick

state: Odisha

address: NIT Rourkela

department: Department of Life Science

country: India

institute: National Institute of Technology

summary: To elucidate potential role of piRNAs in Neuroblastoma (NB), we performed the genome wide profiling in two human NB cell lines, IMR-32 and SH-SY-5Y by adopting high-throughput RNA sequencing (RNA-Seq) and unveil their possible functions in neoplastic pathways. The RNA sequencing results revealed both known and novel piRNAs in both the cell lines. We observed a total 630 annotated mature piRNAs, distributed across chromosomes and mitochondria which were mapped to various genomic locations such as introns, protein coding regions, repeats, pseudogenes, ncRNAs etc. This is the first study reporting the extensive catalogue of human NB piRNAs which will provide a useful resource to dissect complex neoplastic events that are possibly mediated by piRNAs in neuroblastoma. Moreover, these piRNAs could be used as probable small RNA biomarkers for the Neuroblastoma.

overall_design: 1 µg of total RNA isolated from above two cell lines was used as the starting material for library preparation as prescribed by Illumina Truseq small RNA library protocol. The small RNA libraries (size range 16 nts - 40 nts) were sequenced on Illumina NextSeq 500. Upon de-multiplexing, reads are obtained and then analyzed after QC to predict both known and novel piRNAs.

first: Jyoti

last: Roy

first: Bibekanand


title: BRCA1, R-loops and Recombination defects in Ewing's sarcoma (DRIP-seq)

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: Ewing’s sarcoma family of tumors (ESFT) is an aggressive pediatric bone and soft tissue cancer. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the ewing sarcoma break point region 1 (EWSR1) gene, most frequently– EWS-FLI1. We have discovered that loss of EWSR1 leads to accumulation of R-loops, replication stress and impaired homologous recombination, recapitulating breast cancer 1, early onset (BRCA1) deficiency. EWS-FLI1 acts dominant negatively in ESFT to impart the same phenotypes. Further we demonstrate that in ESFT, BRCA1 predominantly associates with the elongating transcription machinery and is unavailable for DNA strand break repair. Gene expression profiling identified upregulated compensatory mechanisms in ESFT cells to process increased R-loops (RNASEH2 and FEN1) and replication stress (Fanconi Anemia). Taken together, our data identifies BRCA1 sequestration due to transcription stress as the mechanistic basis for ESFT chemosensitivity and suggests potential targets for the much lacking second-line therapy.

overall_design: Examination of R-loops was conducted using DRIP-Seq. Three different Ewing's sarcoma cell lines, IMR90 control cell line and U2OS cells transfected with either siRNA against EWSR1 or EWS-FLI1 vector along with appropriate controls were used.Samples were further treated with either vehicle or LD65 dose of etoposide for 6 hours. Genomic DNA was extracted, digested with a restriction enzyme cocktail and used for pulldown of DNA:RNA hybrids.

first: Aparna

last: Gorthi

first: Chen

last: Yidong

first: Alexander

last: Bishop


title: BRCA1, R-loops and Recombination defects in Ewing's sarcoma (RNA-seq)

overall_design: Examination of gene expression of four ESFT cell lines and two control cell lines. Cells were treated to LD65 dose of etoposideand samples collected at 6 hour intervals over 24 hours