title: Genome-wide transcriptomic analysis of CRY2-KO and WT mouse articular cartilage

city: La Jolla

first: Tomas

last: Duffy

address: 10550 N Torrey Pines Rd

institute: TSRI

summary:
OBJECTIVES: Abnormal chondrocyte gene expression promotes osteoarthritis (OA) pathogenesis. RNA-sequencing revealed that circadian rhythm pathway and expression of core clock protein cryptochrome 2 (Cry2) are dysregulated in human OA cartilage. Here we determined expression patterns and function Cry1 and Cry2.
METHODS: Cry mRNA and protein expression was analyzed in normal and OA human and mouse cartilage. Mice with deletion of Cry1 or Cry2 were analyzed for severity of experimental OA and to determine genes and pathways that are regulated by CRY.
RESULTS: In human OA cartilage, CRY2 but not CRY1 staining and mRNA expression was significantly decreased. Cry2 was also suppressed in mice with surgical or aging-related OA. Cry2 KO but not Cry1 KO mice with experimental OA showed significantly increased severity of histopathological changes in cartilage, subchondral bone and synovium. In OA chondrocytes, the levels of Cry1 and Cry2 and the amplitude of circadian fluctuation were significantly lower. RNA-seq on knee articular cartilage of wild-type and Cry2 KO mice identified 53 differentially expressed genes, including known CRY2 target circadian genes Nr1d1, Nr1d2, Dbp and Tef. Pathway analysis indicated that circadian rhythm and extracellular matrix remodeling were dysregulated in Cry2 KO mice.
CONCLUSIONS: These results show an active role of the circadian clock in general, and of CRY2 in particular, in maintaining ECM homeostasis in cartilage. This cell autonomous network of circadian rhythm genes is disrupted in OA chondrocytes. Targeting CRY2 has potential to correct abnormal gene expression patterns and reduce the severity of OA.

overall_design:
We report differentially expressed genes between CRY2-KO and WT mouse articular cartilage samples.
Mouse articular cartilage was collected from both sides of the femoral condyle and tibial plateau from 3-month-old WT and CRY2-KO mice.

first: Martin

last: Lotz


status: Public on Jun 18 2019

city: Braunschweig

first: Claudia

last: Pommerenke

address: Inhoffenstraße 7B

country: Germany

institute: Leibniz Institute DSMZ

type:
Expression profiling by array
Third-party reanalysis

summary: Homeobox genes encode transcription factors regulating basic processes in cell differentiation during embryogenesis and in the adult. Recently, we have reported the NKL-code which describes physiological expression patterns of nine NKL homeobox genes in early hematopoiesis and in lymphopoiesis including main stages of T-, B- and NK-cell development. Aberrant activity of NKL homeobox genes is involved in the generation of hematological malignancies including T-cell leukemia. Here, we searched for deregulated NKL homeobox genes in main entities of T-cell lymphomas comprising peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), hepatospleenic T-cell lymphoma (HSTL), and NK/T-cell lymphoma (NKTL). Our data revealed in all types altogether 19 aberrantly overexpressed genes, demonstrating that deregulated NKL homeobox genes play a significant role in T-cell lymphomas as well. For detailed analyses we focused on NKL homeobox gene MSX1 which is normally expressed in NK-cells and aberrantly activated in T-cell leukemia. This gene was overexpressed in subsets of HSTL patients and HSTL-derived sister cell lines DERL-2 and DERL-7 which served as models to identify mechanisms of deregulation. We performed genomic and expression profiling and whole genome sequencing and revealed mutated and deregulated gene candidates including the fusion gene CD53-PDGFRB exclusively expressed in DERL-2. Subsequent knockdown experiments allowed the construction of an aberrant network involved in MSX1 deregulation containing chromatin factors AUTS2 and H3B/H3.1, PDGF- and BMP-signalling pathways, and homeobox genes NKX2-2 and PITX1. The gene encoding AUTS2 is located at 7q11 and may represent a basic target of the HSTL hallmark aberration i(7q). Our data indicate both oncogenic and tumor suppressor functions of MSX1 in HSTL, reflecting its activity in early lineage differentiation of T- and NK-cells and the presence of NK-cell like characteristics in malignant HSTL cells. In this context, NKL homeobox gene MSX1 may represent a selective target in HSTL tumor evolution. Together, the data highlight an oncogenic role of deregulated NKL homeobox genes in T-cell lymphoma and identified MSX1 as a novel player in HSTL, involved in aberrant NK- and T-cell differentiation.

overall_design:
Deregulated expression of NKL homeobox genes in T-cell lymphomas
Data from 9 published samples were used in this study (IMC-1 and KHYG1 from GEO:GSE19067; NK-92 from GEO:GSE53478; SNK6 from GEO:GSE19067 and 5 T-ALL cell lines i.e. ALL-SIL, CCRF-CEM, PEER, JURKAT, LOUCY from GSE87334)

first: S

last: Nagel

first: C


title: NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs

status: Public on Jun 19 2019

city: Cambridge

first: Lindsey

last: Van Haute

address: Hills Road

department: MRC Mitochondrial Biology Unit

country: United Kingdom

institute: University of Cambridge

type: Methylation profiling by high throughput sequencing

summary: We show that NSUN2 in necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs

overall_design: Cytosine methylation analysis of mammalian mitochondrial enriched RNA


title: Effects of UHMK1 on liver cancer cells

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

institute: University Heidelberg

summary: The impact of the kinase UHMK1 on liver cancer cells is unknown. In order to identify UHMK1-dependent genes, UHMK1 was inhibited by siRNA in comparison to nonsense-siRNA-transfected cells.

overall_design: HepG2 cells were transiently transfected with nonsense/scrambled siRNA or UHMK1-specific siRNA (30 nM). Total mRNA was collected after 24 hours.

first: Kai

last: Breuhahn


title: Point mutations in the PDX1 transactivation domain impair human β-cell development and function

city: Neuherberg

first: Heiko

last: Lickert

address: Ingolstaedter Landstraße 1

department: Institute of Diabetes and Regeneration Research

institute: Helmholtz Zentrum München German Research Center for Environmental Health

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Loss of DKM6A confers drug resistance in acute myeloid leukemia

city: Planegg

first: Helena

last: Domínguez Moreno

state: Bavaria

address: Großhaderner Str. 9

department: Molecular Biology

institute: Biomedical Center of LMU


title: Gene expression-based, through the hybridization of microbial DNA onto the designed probes on microarrys, i.e., GeoChip

city: Beijing

first: Qun

last: Gao

state: Beijing

address: Shuang Qing Road

department: Environmental sicence and engineering

country: China

institute: Tsinghua University

summary: A total of 81515 genes belonging to 31 gene categories were detected in a total of 80 samples

sample_organism:
soil metagenome
compost metagenome
manure metagenome

platform_organism: Bacteria Archaea Eukaryota

overall_design: In the study presented here, microbial functional genes in manure, compost, compost-amended soil and control agricultral soil were detected by GeoChip 5.0 to acquire gene expression profiles profiles of a total of 8,644 unique genes, leading to the successful construction of supervised

first: Jizhong

last: Zhou

first: Xishu


title: TOX Transcriptionally and Epigenetically Programs CD8 T cell Exhaustion

city: Philadelphia

first: Omar

last: Khan

state: PA

address: 421 Curie Blvd. BRB II/III

country: USA

institute: University of Pennsylvania

summary: Transcriptional and epigenetic features were detected in WT versus TOX-/- P14 T cells following 8 days of Cl-13 infection and in in vitro differentiated CD8 T cells or NIH3T3 fibroblasts following retroviral transduction with control or TOX

overall_design: Transcriptional features of WT and TOX-/- P14 T cells following adoptive co-transfer into hosts and subsequent infection with LCMV Cl-13 for 8 days

first: E

middle: J

last: Wherry

first: Sasikanth

last: Manne


title: Point mutations in the PDX1 transactivation domain impair human β-cell development and function (RNA-Seq)

summary: Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes.

overall_design: We performed RNA-seq of control and isogenic PDX1 mutant cell lines at PP stage

first: Xianming

last: Wang

first: Michael

last: Sterr

first: A

last: Ansarullah

first: Ingo

last: Burtscher

first: Anika

last: Böttcher

first: Julia

last: Beckenbauer

first: Johanna

last: Siehler

first: Harald

last: Staiger

first: Hans-Ulrich

last: Häring

first: Gabriele

last: Lederer

first: Thomas

last: Meitinger

first: Filippo

last: Cernilogar

first: Gunnar

last: Schotta

last: Irmler

first: Johannes

last: Beckers

first: Christopher

last: Wright

first: Mostafa

last: Bakhti


title: Point mutations in the PDX1 transactivation domain impair human β-cell development and function (ChIP-Seq)

first: Heiko

last: Lickert

overall_design: We performed ChIP-seq of PDX1 and H3K27ac on PDX1P33T/+ cells at PP stage

last: Wang

first: Martin


title: Expression Profiling of Cisplatin Resistant IGROV1 Cell Lines vs. Wild Type IGROV1 Cell Lines

city: Washington

first: Wenge

last: Zhu

state: DC

address: 800 22nd Street NW, , Science & Engineering Hall 8480

department: School of Medicine and Health Sciences

institute: George Washington University

summary: Ovarian cancer IGROV1 cells were studied to profile the expression of wild type (WT, Cisplatin Susceptible) cells and Cisplatin Resistant (CR) cells. The goal was to gain insights or to build hypotheses into the mechanisms of Cisplatin resistance in this cell-based model.

overall_design: The study included two experimental groups, IGROV1 wild-type cells and IGROV1 Cisplatin resistant (CR) cells.  Each group had two biological replciate samples.

first: Jing

last: Li

first: Wei

last: Sun

last: Zheng


title: Targeting the CDK4/6-Rb pathway enhances response to  PI3K inhibition in PIK3CA mutant lung squamous cell carcinoma

city: Toronto

first: Ming-Sound

last: Tsao

address: 101 College Street, PMCRT 14-401

country: Canada

institute: University Health Network

summary: The purpose of this study is to examine the efficacy of combined PI3K and CDK4/6 inhibition in lung squamous cell carcinoma (LUSC). LUSC patient-derived xenografts (PDX) are used as preclinical models for this study as they are clinically relevant models and have been shown to recapitulate patient tumor hisology, molecular features and drug response. We have demonstrated using the 1x1x1 drug design that PIK3CA mutation predicts response to PI3K inhibitors. This response can be enhanced by combining CDK4/6 inhibitors with PI3K inhibitors, particularly in PIK3CA mutant PDX. PI3K and CDK4/6 inhibitors may be used in combination in PI3K-activated LUSC.

overall_design:
Total RNAs from xenografts were amplified by DASL kit and hybridized to Illumina HT12v4 chip
This dataset consists entirely of untreated xenograft tumors only

first: Ruoshi

last: Shi

first: Shirley

last: Tam

first: Quan


title: Point mutations in the PDX1 transactivation domain impair human β-cell development and function (mRNA microarray)

summary:
Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes.
Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions.

overall_design: We performed Affymetrix microarray analysis on XM001 (GSE106813) and PDX1P33T/+ cells at the pluripotency and PP stages

first: Xianming

first: Michael

last: Sterr

first: Ansarullah

last: Ansarullah

first: Ingo

last: Burtscher

first: Anika

last: Böttcher

first: Julia

last: Beckenbauer

first: Johanna

last: Siehler

first: Harald

last: Staiger

first: Hans-Ulrich

last: Häring

last: Lederer


title: Next Generation RNA Sequencing of Glioblastoma Cancer Associated Fibrobasts

city: San Francisco

first: Jordan

middle: Matthew

last: Spatz

state: CA

address: 1450 3rd Street

department: Department of Neurosurgery

institute: UCSF

summary: Cancer Fibroblasts Isolated from 2 GBM Cases.

overall_design: Methods: GBM CAF RNA libraries were prepared and Illumina HiSeq NGS preformed (UC Davis Dna Technologies Core, Davis, CA) per standard protocols. Differential gene expression, heatmap, and sample cluster were performed by iDEP8.1 (http://bioinformatics.sdstate.edu/idep/).  GBM CAF RNASeq datasets were aligned (BowTie2) and gene exons counted (FeatureCounts) with standard inputs using the Galaxy public server (https://usegalaxy.org/).

first: Manish

last: Aghi


title: Human hepatocellular carcinoma MHCC-97H: Control vs ROCK1 overexpressed vs ROCK2 overexpressed

city: Shanghai

first: HeMing

last: Zhou

state: Shanghai

address: No.100 Haining Road

department: Clinical Pharmacy

institute: Shanghai General Hospital

type:
Expression profiling by array
Non-coding RNA profiling by array

summary:
Transcriptional profiling of human hepatoellular carcinoma (HCC) cell line: MHCC-97H comparing control untreated MHCC-97H cells with MHCC-97H cells overexpressed ROCK1 protein. Goal was to determine the effect of ROCK1 on global MHCC-97H cell mRNA and lncRNA expression.
Transcriptional profiling of human hepatoellular carcinoma (HCC) cell line: MHCC-97H comparing control untreated MHCC-97H cells with MHCC-97H cells overexpressed ROCK2 protein. Goal was to determine the effect of ROCK2 on global MHCC-97H cell mRNA and lncRNA expression.
Transcriptional profiling of human hepatoellular carcinoma (HCC) cell line: MHCC-97H comparing MHCC-97H cells overexpressed ROCK1 with MHCC-97H cells overexpressed ROCK2 protein. Goal was to determine the distinction of ROCK1 & 2 on global MHCC-97H cell mRNA and lncRNA expression.

overall_design: Three-condition experiment, MHCC-97H vs. MHCC-97H-ROCK1 vs. MHCC-97H-ROCK2. Biological replicates: 3 control replicates, 3 ROCK1-overexpressed replicates, 3 ROCK2-overexpressed replicates.

first: Qin

last: Li

first: JiGang

last: Zhang


title: Mapping of CTCF occupancy in interphase and mitosis cells

city: Tsukuba

first: Takeshi

last: SEKIYA

address: 1-1-1 Tennodai

country: Japan

institute: University of Tsukuba

summary: To address whether CTCF is involved in the transcription of early G1 genes immediately after mitotic exit, we carried out ChIP sequencing (ChIP-seq) experiments to evaluate CTCF binding to genomic DNA in asynchronous cells and nocodazole-arrested mitotic cells.  Of the 62,996 peaks identified in the asynchronous cells, 41,397 peaks (65.71%) were lost in the mitotic cells.  However, the 21,599 peaks (34.29%) were identified in the both conditions, indicating that the enrichment level of CTCF is decreased in the mitotic cells.  It has been reported that expression profiles of the first 2 hours of G1 phase in HeLa cells.  A scatter plot comparison of the genes expressed in the early G1 phase indicated the reduced CTCF binding level in the mitotic phase.  These results indicate that CTCF was dissociated or CTCF bound to chromosomes was reduced in the mitotic chromatin and then may be re-associated to chromatin in the early G1 phase.

overall_design: Comparison of CTCF binding sites between asynchronus growing cells and mitotic arrested cells

last: Sekiya

first: Kyosuke

last: Nagata


title: Salt-responsive genes are differentially regulated at the chromatin level between the leaf and root tissues in rice

city: Nanjing

first: Zheng

middle: dong

last: yang

address: weigang NO.1

department: college of agronomy

institute: NJAU


title: Expression data from mouse cerebral cortex

status: Public on Jun 17 2019

city: Suita

first: Yuichiro

last: Oka

state: Osaka

address: 2-2 Yamada-oka

institute: Osaka University

summary:
Cerebral cortex consists of many functional areas that are interconnected via direct axon projections from long association neurons. Long association neurons are located in layers 2/3, 5, and 6b. These neurons are intermingled with callosal neurons, which connect both cerebral hemisphere, in layers 2/3 and 5. To
We used microarrays to compare gene expression profiles of long association neurons and callosal neurons in order to understand genetic programs that specifies these neuronal subtypes.

overall_design: Long association neurons and callosal neuorns in the primary somatosensory area (S1) were labeled by retrograde tracing from the primary motor area (M1) and the S1 on the contralateral hemisphere. 3 days after the tracer injection, mice were perfused with cold PBS, brains were dissected out, and fresh frozed sections were prepared. Labeled neurons were collected one by one with laser microdissection from each layer separately and subjected to total RNA preparation and hybridization on Affymetrix microarrays.

first: Makoto

last: Sato


title: Muscle strain injury exudate favors acute tissue healing and prolonged connective tissue formation in humans

city: Copenhagen

first: Peter

last: Schjerling

address: Bispebjerg Bakke 23, M81

department: Institute of Sports Medicine Copenhagen, ISMC

country: Denmark

institute: Bispebjerg Hospital

summary: Traumatic strain injury in skeletal muscle is often associated with fluid accumulation on the site of rupture, but the role of this injury exudate in cellular responses and healing is unknown. Exudate fluid from subjects suffering from acute hamstering or calf muscle strain injury was aspirated and the cells isolated. These exudate cells and cells derived from the blood were subjected to RNA sequencing and compared to analyze the nature of the exudate cells.

overall_design: Comparison of exudate and blood derived cells from four subjects.


title: Alternaria brassicicola infection regulates miRNAs in Arabidopsis thaliana [mRNA]

city: Oldenburg

first: Sascha

last: Laubinger

address: Carl-von-Ossietzky-Str. 9-11

institute: University of Oldenburg

summary: Plant microRNAs (miRNAs) have been implicated in plant immunity. These mainly focusing Arabidopsis thaliana threatened by (hemi-)biotrophic pathogens such as the bacterial pathogen Pseudomonas syringae. Here, we show that the Arabidopsis miRNA pathway is important for defense responses against the necrotrophic fungus Alternaria brassicicola. The miRNA pathway mutant ago1 exhibits an exaggerated response when treated with A. brassicicola, proposing that AGO1 is positive regulator. We found a subset of Arabidopsis miRNAs that quickly change their expression and their abundance in AGO1 complexes in plants exposed to A. brassicicola. The miRNAs responding to pathogen treatment are mainly targeting genes encoding metabolic enzymes, proteins involved protein degradation or transposons. In case of miR163, A. brassicicola infection results in increased levels of miRNA precursors and preferential accumulation of an unspliced form of pri-miR163, suggesting that A. brassicicola infection changes the transcriptional and post-regulation of pri-miRNAs. miR163 acts as a negative regulator of plant defense because mir163 mutants are more resistant when treated with A. brassicicola. Taken together, our results reveal the existence of positively and negatively acting Arabidopsis miRNA modulating the defense responses against A. brassicicola and highlight the importance of host miRNAs in the interaction between plants and necrotrophic pathogens.

overall_design: mRNA sequencing from untreated, mock treated and Alternaria brassicicola treated plants (3 biological replicates each)

first: Stephanie

last: Rausch

first: Emese

middle: X

last: Szabo


title: Comprehensive characterization of a next-generation antiviral T-cell product and feasibility for application in immunosuppressed transplant patients

status: Public on Jun 16 2019

city: Berlin

first: Karsten

last: Jürchott

address: Luisenstraße

department: Rahel Hirsch Center

institute: BIH at Charité

summary: Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4+ T-cells as well as CD4+ and CD8+ central-memory T-cells (TCM). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin and re-stimulation, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of TCM, partial conversion of other memory T-cell subsets to TCM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post-SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.

overall_design: CMV-specific Tcells derived from fresh blood of buffy coats were expanded with or without the presence of Rapamycin.

first: Leila

last: Amini

first: Michael

last: Schmück-Henneresse


title: Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma (ChIP-seq)

city: Boston

first: Pratiti

last: Bandopadhayay

state: Massachusetts

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary: BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi’s response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the  therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.

overall_design: Drug sensitive and drug tolerant cells were treated with 1uM JQ1 or DMSO controls

first: Rameen

last: Beroukhim


title: Salt-responsive genes are differentially regulated at the chromatin level between the leaf and root tissues in rice [ChIP-Seq]

summary: To elucidate the epigenetic regulation of salt-responsive genes helps to understand the underlying mechanisms that confer salt tolerance in rice. However, it is still largely unknown how epigenetic mechanisms function in regulating the salt-responsive genes in rice and other crops at a global level. In this study, we mainly focused on dynamic changes in transcriptome and histone marks between rice leaf and root tissues during salt treatment by using RNA-seq and ChIP-seq approaches. We demonstrated that the majority of salt-related differentially expressed genes (DEGs) display tissue-dependent changes. Similarly, tissue-dependent chromatin changes have been detected between leaf and root tissues during salt treatment. Most importantly, our study indicates that chromatin states with a combination of marks, rather than an individual mark, most likely play crucial roles in regulating differential expression of salt-responsive genes between leaf and root tissues. Especially, a special CS containing bivalent marks, H3K4me3 and H3K27me3 with a functional exclusion with each other, displays distinct functions in regulating expression of DEGs between leaf and root tissues, H3K27me3-related repressive mark mainly regulates expression of DEGs in root, but H3K4me3-releated active mark dominantly functions in regulation of down-regulated genes and possibly antagonize the repressive role of H3K27me3 in up-regulated genes in leaf. Thus, our findings indicate salt-responsive genes are differentially regulated at the chromatin level between the leaf and root tissues in rice, which provides new insights in the understanding of chromatin-based epigenetic mechanisms that confer salt tolerance in plants.

overall_design: Examination of six different histone modifications in 2 tissue, each tissue have two diifferent growth conditions

first: Dongyang

last: Zheng

first: Lei

last: Wang

first: Xiucai

last: Pan

first: Wenli


title: Cadmium exposure induces SNAIL1 expression via downregulation of miR-30e in human bronchial epithelial cells

city: Duarte

first: Rama

last: Natarajan

state: California

address: 1500 E Duarte Rd

department: Department of Diabetes Complications and Metabolism

institute: Beckman Research Institute, City of Hope

summary:
Purpose: To understand the molecular mechanisms underlying Cd exposure-induced diseases.
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 Cd exposure,  0, 2.5, 5 and 10 µM of CdCl2 was added to the media and the cells were cultured for 72h.
Results: This study shows that cadmium exposure induces SNAIL1 expression via miR-30e  downregulation and the cells undergo epithelial-mesenchymal transition.

overall_design: RNA Seq gene expression comparision done in replicates

first: Suresh

last: Cuddapah

first: Vinay

last: Singh Talwar

first: Cynthia

last: Jose

first: Lakshmanan

last: Jagannathan


title: CREB mediates transient and sustained genomic responses to cAMP via distinct mechanisms [RNA-seq]

city: La Jolla

first: Sam

last: Van de Velde

address: 10010 North Torrey Pines Road

department: PBL

institute: Salk Institute

summary: We investigated genome-wide occupancy of CREB, CREB coactivators, lineage determining transcription factors and histone acetylation to uncover mechanisms behind tissue-specific gene induction by cAMP in pancreatic islets. CREB mediates effects of cAMP on cellular gene expression. Most core CREB target genes are ubiquitously induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell genes by binding to sites within distal enhancers. By contrast with its transient effects on core target genes, CREB stimulates pancreatic beta cell specific gene expression in a sustained manner, reflecting increases in the CBP-mediated acetylation of resident nucleosomes that recruit the chromatin reader BRD4. CREB cooperates with the lineage specific activator Neurod1 in establishing cAMP-responsive enhancers in beta cells. As deletion of a CREB-Neurod1 bound enhancer within the Lrrc10b-Syt7 super-enhancer locus disrupted the expression of both genes and decreased glucose-induced insulin secretion, our results demonstrate how cooperativity between signal dependent and lineage determining factors promotes the expression of cell type-specific gene programs in response to extracellular cues.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: RNA-seq was performed in primary mouse islets and hepatocytes as well as rat pancreatic cell lines.


title: Loss of DKM6A confers drug resistance in acute myeloid leukemia (ChIP-seq of AML cell lines K562 and THP-1)

first: Helena

last: Domínguez Moreno

summary: Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1). Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

overall_design: H3K27ac ChIP-seq of AML cell lines K562 and THP-1

first: Sophie

middle: M

last: Stief

first: Anna-Li

last: Hanneforth

first: Raphael

last: Mattes

first: Sabrina

last: Weser

first: Michela

last: Carlet

first: Wen-Hsin

last: Liu

middle: D

last: Bartoschek

first: Matthias

last: Oettle

first: Binje

last: Vick

first: Bianka

last: Ksienyzk

first: Julia

last: Kempf

first: Belay

last: Tizazu

first: Maja

last: Rothenberg-Thurley

first: Hilmar

last: Quentmeier

first: Wolfgang

last: Hiddemann

first: Sebastian

last: Vosberg

first: Philipp

last: Greif

first: Klaus

middle: H

last: Metzeler

first: Gunnar

last: Schotta

last: Bultmann

first: Irmela

last: Jeremias

first: Heinrich

last: Leonhardt

last: Spiekermann


title: Zika virus antagonizes interferon response in patients and disrupts RIG-I-MAVS interaction through its CARD-TM domains

city: GUANGZHOU

first: zhiyong

last: shen

address: North Campus, Sun Yat-Sen University, No. 74, 2nd Yat-Sen Road/Street Yuexiu District

department: Department of Microbiology

institute: Sun Yat-sen University

summary: The emerging threat to global health associated with the Zika virus (ZIKV) epidemics and its link to severe complications highlights a growing need to better understand the pathogenic mechanisms of ZIKV.  Accumulating evidence for a critical role of type I interferon (IFN-I) in protecting hosts from ZIKV infection lies in the findings that ZIKV has evolved various strategies to subvert the host defense line by counteracting the early IFN induction or subsequent IFN signaling.  Yet, mechanisms underlying the counter-IFN capability of ZIKV and its proteins, which might contribute to the well-recognized broad cellular tropisms and persistence of ZIKV, remain to be fully understood.  In our current study, using RNA sequencing-based transcriptional profiling from the whole blood cells isolated from patients acutely infected by ZIKV, we found that transcriptional signatures of antiviral interferon-stimulated genes and innate immune sensors was absent in ZIKV-infected patients presents inactive as compared to healthy donors, suggesting that ZIKV might suppress the induction of IFN-I during the natural infection process in human.  Furthermore, utilizing cellular or extracellular analysis of molecular interaction in a ZIKV NS4A-overexpression system, or in the context of actual ZIKV infection, we have identified that ZIKV NS4A directly binds MAVS and thereby interrupts RIG-I/MAVS interaction through its CARD-TM domains, leading to attenuated production of IFN-I.  Taken together, these findings originated from patient studies have added new knowledge and molecular details to our understanding regarding how ZIKV mediates suppression of the IFN-I system and may provide new basis for future development of anti-ZIKV strategies.

overall_design: Peripheral blood were collected from three patients during the onset period and also from three healthy donors by using the anticoagulant vacuum blood collection tubes (BD Franklin Lakes, NJ), and subsequently whole blood cells were collected by centrifugation at 800 x g for 20 min at room temperature.  Total RNA was extracted from the collected blood cells with the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction.   Host cell transcriptional profiling was performed using RNA deep sequencing by employing the Annoroad Gene Technology Co., Ltd (Beijing, China).

first: Gucheng

last: Zeng

first: Mengfeng

last: Li

first: Xun

last: Zhu


title: Transcriptome alterations in porcine adipocytes downstream of TLR-2, -3 and -4 ligation

city: Sendai

first: Haruki

last: Kitazawa

state: Miyagi

address: 468-1 Aramaki Aza Aoba, Aoba-ku,, Sendai

institute: Tohoku University

summary: Adipocytes are functionally the most important cell type in adipose tissue, which play a key role in systemic metabolism and inflammation. Toll-like receptors (TLRs) are important innate immune receptors involve in the early recognition of infection flowed by inflammatory responses. We recently reported that nine members of TLR (1-9) are expressed in the porcine adipocyte. To further development of our in vitro adipocyte model for evaluating potential immunobiotics, herein we characterized the global transcriptome alterations in porcine mature adipocytes (PMA) following stimulation with Pam3csk4, Poly(I:C) and LPS for 12 h, as a specific ligand for TLR2, TLR3 and TLR4, respectively. Analysis of microarray data revealed that 530 (218 up, 312 down), 520 (245 up, 275 down) and 525 (239 up, 286 down) genes were differentially expressed in PMA following stimulation with Pam3csk4, poly (I:C) and LPS stimulation, respectively. Gene ontology classification revealed that differentially expressed genes (DEGs) are involved in several biological process including immune response, inflammatory responses, cytokine-mediated signaling, chemokine-mediated signaling. The accurately annotated genes were organized into two: immune response related genes (cytokine, chemokine, complement factors, adhesion molecules and signal transduction), and genes involved with metabolic and endocrine function (hormones and receptors, growth factors and lipogenic). We inferred protein interaction networks from common DEGs and found that EGR1, NOTCH, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10 and C3 are the major hub genes of TLR induced transcriptional network of porcine adipocyte. Potential hubs of protein interaction network also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Expressions of the important genes were also measured by qRT-PCR, confirming the microarray results. Our results provide new insights of transcriptome modifications of adipocytes association with immune response, metabolic and endocrine function.

overall_design: TLRs induced gene expression in porcine adipocyte was measured after 12 h exposure of Pam3csk, Poly (I:C) and LPS as a ligand for TLR2, TLR3 and TLR4, respectively. Single sample was generated in each of three cases along with a non-stimulated control.

first: Igata

last: Manami

first: Islam

last: Aminul

first: Albarracin

last: Leonardo

first: Villena

last: Julio

first: Kitazawa

last: Haruki


title: Loss of DKM6A confers drug resistance in acute myeloid leukemia (ChIP-seq of AML cell lines MM-1 and MM-6)

city: Planegg

first: Helena

last: Domínguez Moreno

address: Großhaderner Str. 9

institute: Biomedical Center of LMU

overall_design: H3K27ac and H3K27me3 ChIP-seq of AML cell lines MM-1 and MM-6

first: Sophie

last: Stief

first: Anna-Li

last: Hanneforth

first: Raphael

last: Mattes

first: Sabrina

last: Weser

first: Michela

last: Carlet

first: Wen-Hsin

last: Liu

first: Michael

last: Bartoschek

first: Matthias

last: Oettle

first: Binje

last: Vick

first: Bianka

last: Ksienyzk

first: Julia

last: Kempf

first: Belay

last: Tizazu

first: Maja

last: Rothenberg-Thurley

first: Hilmar

last: Quentmeier

first: Wolfgang

last: Hiddemann

last: Vosberg

last: Greif

first: Karsten


title: RB1 deletion in RB-pathway disrupted cells results in DNA damage and cancer progression

city: London

first: Fred

last: Dick

state: ON

address: Cancer Research Labs 790 Commissioners Road East

institute: Western University

summary: Proliferative control in cancer cells is frequently disrupted by mutations in the RB-pathway. Paradoxically, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB-pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage in CDKN2A deleted cancer cells when RB1 mutations are induced. We created isogenic RB1 wild type, heterozygous, and null genotypes. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immune compromised mice RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the pre-existing proliferative control deficit.

overall_design: γH2AX localization was studied in three different genotypes of U2OS osteosarcoma cells: RB1+/+, RB1+/- and RB1-/-. As a control for possible histone density differences, histone 4 (H4) ChIP-Seq was also performed.

first: Frederick

middle: A

first: Aren

middle: E

last: Marshall


title: Specific contributions of cohesin-SA1 and cohesin-SA2 to TADs and Polycomb domains in embryonic stem cells.

city: Madrid

first: Daniel

last: Gimenez

address: Calle de Melchor Fernández Almagro, 3

country: Spain

institute: CNIO

summary: Cohesin complex, a main organizer of mammalian genomes, exists in two versions that differ in the identity of the STAG/SA subunit, which can be SA1 or SA2. Mouse embryonic stem cell (mESC) provide a useful system to address the specific contributions of each variant to genome architecture and gene expression, since 3D organization of super- enhancers and Polycomb domains is essential to achieve transcription of pluripotency factors and repression of lineage specification genes, respectively. Hi-C analyses reveal That cohesin-SA1 preserves the integrity of topological associating domains (TAD) boundaries together with CTCF and prevents excessive segregation of same-class Compartment regions. Cohesin-SA2 is enriched within super-enhancers and Polycomb domains. Moreover, it contributes to establishment and compaction of Polycomb domains through PRC1 recruitment thereby promoting interchromosomal interactions among Hox gene promoters and favoring gene repression. In contrast, these interactions are counteracted by cohesin-SA1. The opposite effects of the two complexes on genome topology explain the distinct consequences of their ablation for the mESCs Transcriptome.

overall_design: ChIP-seq for cohesin subunits was performed in mES cells growing under serum/LIF (SA1, SA2 and SMC1) or serum/LIF/2i (SA1 and SA2) conditions, and peak calling was done against the corresponding input sample. Suz12 and Ring1B ChIP-seq was performed in duplicates in serum/LIF growing mES control and depleted from SA1 or SA2 by means of Dharmacon SMART pool siRNAs. RNA-seq was performed in triplicates in serum/LIF growing mES control and depleted from SA1, and in duplicates from SA2 depleted cells by means of Dharmacon SMART pool siRNAs. HiC-seq was performed in two independent biological replicates in mES cells growing under serum/LIF/2i or serum/LIF mES cells control and depleted from SA1 and SA2 by means of Dharmacon SMART pool siRNAs.

first: Ana

last: Cuadrado

last: Giménez-Llorente

first: Aleksandar

last: Kojic

first: Miriam

last: Rodrígez-Corsino

first: Yasmina

last: Cuartero

first: François

last: Le Dily

first: Marc

last: Marti-Renom

last: Losada


title: Cytokeratin 18 knockdown decreases cell apoptosis by regulating the transcription and alternative splicing in Hela cell

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

summary: Cytokeratin 18(CK18), one of the major components of intermediate filaments (IF) in simple-type epithelial cells, has been used as the biomarker of several cancers. CK18 has been also reported to be dysregulated in cervical cancers, however, the precise molecular pathways regulated by CK18 remained elusive. In this study, CK18 was knockdown in Hela cells and which led to a significantly decrease of cell apoptosis compared with control cells. The global transcript profile and alternative splicing of CK18 knockdown cells and control were analyzed. A total of 593 differentially expressed genes were identified, and these genes were mainly involved in synaptic transmission, immune response, innate immune response, signal transduction pathways. Strikingly, hundreds of genes were regulated by CK18, which mainly enriched in immune and apoptosis related pathways. In conclusion, our study indicates that CK18 plays important regulatory role in cervical cancers in transcriptional level as well as alternative splicing regulation.

overall_design: transcrptional analysis of CK18 knockdown and control in Hela cells

first: Yi

last: CHENG


title: Screening an alternative flame retardant using biological and transcriptomic endpoints in fish embryos

city: Durham

first: Weichun

last: Huang

state: NC

address: 109 TW Alexander Dr

department: ORD

institute: US EPA

summary: Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. By design, environmentally-high exposures of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite, or observed for development and survival through larval stage. Global transcript profiling, which requires no a priori information, was applied using a large number of embryos (16) per treatment to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals, and does not present a unique gene expression pattern. More broadly, this study advances the usefulness of a biological testing and analysis system that complements current screening methods to characterize chemicals of ecological and human health concern

overall_design: one control and two treatment groups: low and high concentration, each with 16 biological replicates

first: David

middle: C

last: Bencic

first: Robert

middle: L

last: Flick

first: Diane

last: Nacci

first: Bryan

middle: W

last: Clark

first: Lawrence

last: Burkhard

first: Tylor

last: Lahren

first: Adam

last: Biales


title: A systematic reassessment of N6-methyladenine in metazoan genomes

first: Eric

last: Greer

state: MA

address: 320 Longwood Avenue

department: Newborn Medicine/Department of Pediatrics

institute: Boston Children's Hospital/Harvard Medical School

summary: We report the application of SMRT seqeuncing from Pacific Biosciences to study the DNA base modifications in dam-/dcm- and wild type E.coli.

overall_design: Examination the 4mC and 6mA modifcation in dam-/dcm- and wild type E.coli

first: Simon

middle: Y

last: Wang


title: Calmodulin-binding transcription activator (CAMTA) 6 is a key regulator of Na+ homeostasis and salinity stress responses during early germination

city: Tel Aviv

first: Metsada

last: Pasmanik-Chor

address: Ramat Aviv

department: Biology

country: Israel

institute: Tel Aviv University

summary: In view of the continuous salinization of arable lands world-wide, there is an urgent need to better understand the mechanisms underlying plant responses to salt stress at different stages of their development. We investigated the role of calmodulin (CaM)-binding transcription activator 6 (CAMTA6) under salinity stress during early germination in Arabidopsis. These analyses suggest that ABA signaling is involved in CAMTA6-dependent salt-responsive gene expression, consistent with the ABA hyper-tolerance phenotype and the lack of HKT1 response to ABA and NaCl in the camta6 mutants.

overall_design: In order to study the germination behavior of camta6 mutants in the presence of salt (NaCl), seeds of wild-type (Col-0) and an allele of camta6 mutant (camta6-5) with 0 (control) or 200 mM NaCl.

first: Doron

last: Shkolnik

first: Aliza

last: Finkler

first: Hillel

last: Fromm


title: SULT2B11b regulates sensitivity to TNF-mediated cell death in Prostate Cancer

city: West Lafayette

first: Nadia

middle: Marie

last: Atallah

state: IN

address: 201 S. University St

department: Center for Cancer Research

institute: Purdue University

summary: Single-cell mRNA sequencing (scRNA-seq) study was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus non-targeting (Control) KD LNCaP cells.  Over 2,000 differentially expressed (DE) genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway.

overall_design: To prepare for single-cell analysis, 2x10^5 LNCaP cells were plated in a 12-well plate coated with poly-L-lysine and allowed to adhere for 24 hours.  LNCaP cells were transfected with non-targeting (control) siRNA (Dharmacon) or SULT2B1 siRNA (IDT, HSC.RNAI.N004605.12.2), previously validated to knockdown SULT2B1b expression and function in prostate cancer cells (Figure 3.4E).234  48 hours following siRNA transfection, cells were harvested with trypsin and stained with Zombie Violet viability dye (Biolegend, 423114) according to the manufacturer’s instructions.

first: Renee

last: Vickman

first: Jiang

last: Yang

first: Gregory

last: Cresswell

first: Faye

last: Zhen

first: Chi

last: Zhang

first: Rebecca

last: Doerge

first: Scott

last: Crist

first: Andrew

last: Mesecar

first: Timothy

last: Ratliff


title: A multiscale map of the stem cell state in pancreatic adenocarcinoma

city: Glasgow

first: Neil

last: Robertson

state: Scotland

address: Switchback Road

department: Beatson Institute for Cancer Research

institute: University of Glasgow

summary: Therapy resistance and resultant relapse remain key challenges in pancreatic cancer, and are in part driven by the inherent heterogeneity of the tumor that prevents effective targeting of all malignant cells. Here we utilized a combination of RNA-seq, ChIP-seq and genome-wide CRISPR screening to systematically map the dependencies of pancreatic cancer stem cells, highly drug resistant cells that are also enriched in the capacity to drive progression 1-4 . Integration of these data revealed an unexpected role for immuno-regulatory genes in stem cell self-renewal and maintenance in KP f/f C tumors. In particular, Retinoic acid receptor-related orphan receptor gamma (ROR g) , a nuclear hormone receptor known for its role in inflammatory cytokine responses and T cell differentiation 5,6 , emerged as a key regulator of stem cells. ROR g expression rose with pancreatic cancer progression, and its inhibition via genetic approaches or small molecule inhibitors led to a striking defect in pancreatic cancer growth in vitro and in vivo. Collectively, these data reveal an unexpected co-option of immuno-regulatory signals by pancreatic cancer stem cells, and suggest that therapeutics currently being used for autoimmune indications should be considered for treatment of pancreatic cancer.

overall_design: Examination of the histone modification H3K27ac in REM2+/EpCAM+ (stem) and REM2-/EpCAM+ (non-stem) cells.

first: Nikki

last: Lytle

first: L

middle: P

last: Ferguson

first: Ray

last: Fox

first: Kathryn

last: Gilroy

first: Peter

last: Adams

first: Tannishtha

last: Reya


title: The MybMuvB complex is required for YAP dependent transcription of mitotic genes

city: Wuerzburg

first: Stefan

last: Gaubatz

address: Am Hubland

department: Biochemistry and Cell Biology

institute: University of Wuerzburg

summary: YAP/TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin-association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes co-activated by YAP and B-MYB is associated with poor survival of cancer patients. Together, our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
ChIP-seq of LIN9, YAP, B-MYB and histone marks in murine lung adenocarcinoma cells (KPL) and in human MCF10A-YAP5SA cells.
Gene expression changes upon Lin9-depletion in KPL cells were measured with RNA-seq.

first: Grit

last: Pattschull

first: Susanne

last: Walz

first: Eva

last: Ruehl

first: Elmar

last: Wolf

first: Carsten

last: Ade

last: Kneitz

first: Andreas

last: Rosenwald

first: Bjoern

last: von Eyss


title: Adaptive Responses of Shewanella decolorationis to the Toxic Organic Extracellular Electron Acceptor in Anaerobic Respiration

city: Guangzhou

first: Yun

last: Fang

state: Guangzhou

address: 100 Central Xianlie Road

institute: Guangdong Institute of Microbiology

summary: Bacterial anaerobic respiration using extracellular electron acceptor plays a predominant role in global biogeochemical cycles. However, the bacterial adaptive mechanisms to the toxic organic pollutant as the extracellular electron acceptor during anaerobic respiration is not clear, which limits us to optimize the strategies for the bioremediation of contaminated environment. Here, we report the physiological characteristics and the global gene expression of an ecologically successful bacterium Shewanella decolorationis S12 when using a typical toxic organic pollutant, amaranth, as the extracellular electron acceptor. Our results revealed that filamentous shift (the cells stretched to fiber-like shapes as long as 18 μm) occurred under amaranth stress. Persistent stress led to higher filamentous cell rate and decolorization ability in subcultural cells compared with parental strains. Additionally, the expression of genes involved in cell division, chemotaxi system, energy conservation, damage repair, and material transport in filamentous cells were significantly stimulated. The detailed roles of some genes with significantly elevated expressions in filamentous cells were identified by site-directed mutagenesis, such as the outer membrane porin genes ompA and ompW, the cytochrome C genes arpC and arpD, the global regulatory factor gene rpoS and methyl-accepting chemotaxis proteins genes SHD_2793 and SHD_0015. Finally, a conceptual model was proposed to help deepen our insights into both the bacterial survival strategy when toxic organics were present, and the mechanisms in which these toxic organics were biodegraded as the extracellular electron acceptors.

overall_design: mRNA profiles of the normal (exposed to azo dye for 0.5h) and filamentous (exposed to azo dye for 8h) cells of Shewanella decoloriationis S12 were generated by deep sequencing, in triplicate, using Illumina Hiseq 2000.

first: Meiying

last: Xu


title: Assessing chromatin accessibility in WT and STAT4-/- ILC1s

city: Birmingham

first: Laurie

last: Harrington

state: AL

address: 845 19th street south, BBRB 467

department: CDIB

institute: University of Alabama at Birmingham

summary: STAT4 dynamically interacts with the genomic landscape and functions in modulating chromatin accessibility to influence gene expression.  We aimed to evaluate the impact of STAT4 on chromatin accessibility of colonic lamina propria ILC1s. To address this aim, we sorted ILC1s from the colonic LP of unmanipulated C57BL/6 and STAT4-/- mice and performed the assay for transposase-accessible chromatin using sequencing (ATAC-seq).

overall_design: Chromatin accessibility profiles of ILC1s isolated from the colonic lamina propria of WT and STAT4-/- mice, in duplicate.

first: Sarah

last: Dulson

first: David

last: Crossman

first: Emily

last: Watkins


city: Cambridge

first: Gabriela

last: Alexe

address: 415 Main St.

department: Computational Biology and Bioinformatics

institute: Broad Institute

summary:
Exploiting an Asp-Glu “switch” in glycogen synthase kinase 3 to design paralog selective inhibitors for use in acute myeloid leukemia: Genome-wide transcriptional profile for the GSK3β selective inhibitor BRD3731.
Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the WNT pathway, remains a therapeutic target of interest in many diseases. While dual GSK3α/β inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, are a concern in the translation of this target class to cancer therapy, particularly for the treatment of acute myeloid leukemia (AML). Knockdown of GSK3α or GSK3β individually does not increase β-catenin in certain cellular subtypes and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, only inadequate chemical tools exist.  The design of selective ATP competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity, 100% similarity) in their ATP binding domains. Taking advantage of an Asp133®Glu196 “switch” in their hinge binding domains, we present a rational design strategy towards the discovery of a paralog selective set of GSK3 inhibitors. These first-in-class GSK3α and GSK3β selective inhibitors provided insights into GSK3 targeting in AML where GSK3α has been identified as a therapeutic target using genetic approaches. Our GSK3α selective compound (BRD0705) inhibits kinase function and does not stabilize β-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells while no effect is observed on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies validate feasibility of paralog selective GSK3α inhibition offering a promising therapeutic approach in AML.

overall_design: Genome-wide transcriptional profile for the GSK3β selective inhibitor BRD3731 in U937 cells.

first: Kimberly

last: Stegmaier


title: Profiling the transcriptome of murine radial glial and astrocyte precursor cells

city: Indianapolis

first: Jonathan

last: Wilson

state: INDIANA

address: 723 W. Michigan St.

institute: IUPUI

summary:
The goal of this study is to evaluate transcriptome profiles that accompany mouse radial glial cells and astrocytes isolated from the developing central nervous system.
GLAST-positivity, a marker of radial glia and astrocyte lineage, was exploited to isolate cells from postnatal day 0 (P0) and P8 mouse brains using GLAST-specific antibodies conjugated to magnetic beads. mRNA from four separate biological replicates of GLAST-positive cells for each postnatal stage were used to construct cDNA libraries following TruSeq RNA sample preparation. Sequencing of libraries was conducted on Illumina HiSeq2000 using 100bp paired-end reads with 25 million reads per sample. Sequenced reads were aligned to the mouse genome (mm9) with the TopHat workflow and fragments per kilobase of transcript per million (FPKM) was calculated. Differential expression was determined using EdgeR with continuous dispersion and false discovery rate was calculated.
RNA-seq data confirmed appropriate expression of 45 known housekeeping genes from different cell types in both P0 and P8 post-natal mouse brain. Differential expression of 1438 genes at P0 and 1100 at P8 GLAST-positive cells were found to have a fold change ≥2 and FDR <0.05. 18 genes were confirmed with qRT-PCR, demonstrating the validity of the RNA-seq method.

overall_design: mRNA profiles of P0 and P8 wild type (WT) C57Bl6/J GLAST-postive mouse brain cells were generated, in quadruplicate.

first: Teri

last: Belecky-Adams


title: CaMKK2 in myeloid cells is a key regulator of the immune suppressive microenvironment in breast cancer

first: LUIGI

last: RACIOPPI

state: North Carolina

address: La Salle, GSRB1 2006

department: Medicine

institute: Duke University

summary: Tumor-associated myeloid cells play a pivotal role in the regulation of processes that control tumor growth and metastasis, and their accumulation in tumors is an established negative prognostic factor in breast cancer. Here, we show that inhibition of calcium/calmodulin-dependentkinase kinase 2 (CaMKK2) expression within myeloid cells inhibits tumor growth in mouse models of mammary cancer. This activity is associated with the accumulation of macrophages within the tumor microenvironment that express high levels of the major histocompatibility molecule class II molecule I-A (MHC II I-A), and granzyme positive CD8+T-cells. Treatment with specific   CaMKK2 inhibitors block tumor growth, in a CD8+ T-cell dependent manner, and facilitates favorable reprogramming of the immune cell microenvironment. CaMKK2 protein expression was highest in the most aggressive subtypes of human breast cancer and, in the most malignant tumors, both tumor cells and tumor-associated macrophages express high levels of this enzyme. In aggregate, these findings implicate CaMKK2 as a macrophage-selective immune checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.

overall_design: Tumor-conditioned medium E0771 mouse breast cancer cell lines were cultured in RPMI-1640 (Gibco, MA, USA) supplied with 2 mM L-glutamine, 1.5g/L sodium bicarbonate, 1.0 mM sodium pyruvate (all from Gibco, MA, USA) and 8% fetal bovine serum (FCS, Hyclone, MA, USA). Cells were maintained in a humidified 37°C CO2 incubator and passed at 70% confluence. To generate conditioned medium, medium was removed from 70% confluent cultures and replaced with fresh RPMI-1640 8% FBS. After 3 days, culture supernates were collected and aliquots stored at -80°C. The protocol used for generating BMDM has been described previously (Racioppi et al. 2012). Briefly, hind limb bones were removed from mice and crushed in a mortar with 5 ml of Hanks' balanced salt solution (Mediatech, Manassas, VA) plus 2% FBS (Gemini Bio-Products, West Sacramento, CA) supplemented with 2 mM EDTA. Bone marrow (BM) cell suspensions were passed through a 70 μm strainer (BD Falcon) and stratified on Lympholyte (Cedarlane, Burlington, NC). The low-density fraction containing BM nucleated cells was collected, and the concentration was adjusted to 2 × 10^6 cells/ml in complete medium (10% fetal bovine serum DMEM, high glucose, supplemented with glutamine, pyruvate, and HEPES, no-phenol red) containing 30% L929-conditioned medium (differentiation medium, RM). Subsequently, BM cells were cultured for 5 days in Corning® Costar® Ultra-Low attachment multi-well plates (Sigma). To establish the purity of BMDM, cells were double-stained with anti-CD11b and anti-F4/80 antibodies. Based on this analysis, more than 95% of cells co-expressed these markers, which is a characteristic of macrophages. In the same experiments, tumor-conditioned medium (20% v/v) was added to differentiation medium. BMDM were generated in the presence of RM or CM from WT and 16 Camkk2-/- mice. Four biological replicates for each genotype were generated using long bones of 16 WT and 16 Camkk2-/- mice. Microarray analysis was performed at Sequencing and Genome Technologies Shared Resource (Duke University).

first: Luigi

last: Racioppi

first: Wei

last: Huang

first: Scott

last: Lawrence

first: William

last: Lento

first: Anna

last: Masci

first: Yiqun

last: Jiao

first: Sunghee

last: Park

first: Brian

last: York

first: Yaping

last: Liu

first: Amy

last: Baek

last: Drewry

last: Zuercher

first: Joseph

last: Geradts

first: Allison

last: Hall

first: Anthony

middle: R

last: Means

first: Nelson

last: Chao

first: Donald

last: McDonnell


title: Alternaria brassicicola infection regulates miRNAs in Arabidopsis thaliana

city: Oldenburg

first: Sascha

last: Laubinger

address: Carl-von-Ossietzky-Str. 9-11

institute: University of Oldenburg

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Alternaria brassicicola infection regulates miRNAs in Arabidopsis thaliana [sRNA]

summary: Plant microRNAs (miRNAs) have been implicated in plant immunity. These mainly focusing Arabidopsis thaliana threatened by (hemi-)biotrophic pathogens such as the bacterial pathogen Pseudomonas syringae. Here, we show that the Arabidopsis miRNA pathway is important for defense responses against the necrotrophic fungus Alternaria brassicicola. The miRNA pathway mutant ago1 exhibits an exaggerated response when treated with A. brassicicola, proposing that AGO1 is positive regulator. We found a subset of Arabidopsis miRNAs that quickly change their expression and their abundance in AGO1 complexes in plants exposed to A. brassicicola. The miRNAs responding to pathogen treatment are mainly targeting genes encoding metabolic enzymes, proteins involved protein degradation or transposons. In case of miR163, A. brassicicola infection results in increased levels of miRNA precursors and preferential accumulation of an unspliced form of pri-miR163, suggesting that A. brassicicola infection changes the transcriptional and post-regulation of pri-miRNAs. miR163 acts as a negative regulator of plant defense because mir163 mutants are more resistant when treated with A. brassicicola. Taken together, our results reveal the existence of positively and negatively acting Arabidopsis miRNA modulating the defense responses against A. brassicicola and highlight the importance of host miRNAs in the interaction between plants and necrotrophic pathogens.

overall_design: small RNA sequencing from untreated, mock treated and Alternaria brassicicola treated plants (3 biological replicates each).  AGO1-immunoprecipitated sRNAs also sequenced.

first: Stephanie

last: Rausch

first: Emese

last: Szabo


title: CREB mediates transient and sustained genomic responses to cAMP via distinct mechanisms

city: La Jolla

first: Sam

last: Van de Velde

address: 10010 North Torrey Pines Road

institute: Salk Institute


title: lncRNA expression profile in CRC

status: Public on Jun 15 2019

first: Dong-liang

last: Chen

address: No.651 Dong Feng East Road

institute: Sun Yat-sen University Cancer Center

summary: The purpose of this study is to identify lncRNAs involved in the pathology of colorectal cancer (CRC) progression and investigate their underlying mechanisms. The differentially expressed lncRNAs were identified between 15 CRC tissues and 15 adjacent normal tissues by Arraystar lncRNA microarrays

overall_design: 15 CRC tissues and 15 adjacent normal tissues were subjected to microarray to select differentially expressed lncRNAs

first: D

first: R

last: Xu


title: Transcriptome analysis of drought treated Caragana korshinskii and control

status: Public on Jun 14 2019

city: Beijing

first: Yan

last: Long

state: China

address: Zhongguancun South street No.12

institute: Chinese Academy of Agricultural Sciences

summary: To discover the molecular events that occur during exposure to drought, two digital gene expression (DGE) libraries were constructed by sequencing RNA extracted from two pools of control and drought-treated plants using Illumina technology.

overall_design: One month old seedlings were used as materials to do drought treatment, and each sample was with two replicates


title: Liver lncRNA gene expression in response to CAR/PXR agonists

city: Boston

first: David

middle: J.

last: Waxman

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

summary: Gene expression in livers of male and female mice treated with the CAR agonist ligand TCPOBOP or the PXR agonist ligand PCN

overall_design: RNA-Seq was performed on liver RNA isolated from individual mice 3 hr or 6 hr after treatment with TCPOBOP or PCN. The corresponding untreated mice livers were included in GEO series GSE66140, namely GSM1612431 (liver G81_M1) as control for G81_M3 and G81_M5; GSM1612432 (liver G81_M2) as control for G81_M4 and G81_M6; GSM1612433 (liver G81_M7) as control for G81_M9 and G81_M10; samples GSM1612759-GSM1612761 for the G86 series; and samples GSM1612774-GSM1612776 for the G95 series.

first: Nicholas

last: Lodato

first: Tisha

last: Melia


title: CREB mediates transient and sustained genomic responses to cAMP via distinct mechanisms [ChIP-seq]

state: CA

summary: We investigated genome-wide occupancy of CREB, CREB coactivators, lineage determining transcription factors and histone acetylation to uncover mechanisms behind tissue-specific gene induction by cAMP in pancreatic islets. CREB mediates effects of cAMP on cellular gene expression. Most core CREB target genes are ubiquitously induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell genes by binding to sites within distal enhancers. By contrast with its transient effects on core target genes, CREB stimulates pancreatic beta cell specific gene expression in a sustained manner, reflecting increases in the CBP-mediated acetylation of resident nucleosomes that recruit the chromatin reader BRD4. CREB cooperates with the lineage specific activator Neurod1 in establishing cAMP-responsive enhancers in beta cells. As deletion of a CREB-Neurod1 bound enhancer within the Lrrc10b-Syt7 super-enhancer locus disrupted the expression of both genes and decreased glucose-induced insulin secretion, our results demonstrate how cooperativity between signal dependent and lineage determining factors promotes the expression of cell type-specific gene programs in response to extracellular cues.

overall_design:
Cells were fixed in 0.75% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min. Frozen cell pellets were resuspended in buffer LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-laurylsarcosine, protease inhibitor cocktail, Sigma P9599) and sonicated (Active Motif EpiShear Probe Sonicator). After sonication, Triton-X100 was added to the extract (1% final) and chromatin was cleared by centrifugation at 17,000 g for 10 min. 35 μl protein A agarose beads (Thermo Scientific 20333) were washed twice with PBS + 0.1% BSA and coupled with 3 ug antibody for 4 hours in PBS + 0.1% BSA at 4°C with rotation. 500 μl extract was added to antibody coupled beads and incubated overnight at 4°C with rotation. Beads were washed three times in 500 μl wash buffer 1 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) and three times in wash buffer 2 (20 mM Tris-HCl pH 7.4, 250 mM LiCl, 1 mM EDTA, 1% Triton X-100, 0.7% Na-deoxycholate). Elution was then performed by incubating beads in 50 μl elution buffer 1 (TE, 1% SDS) for 15 min at 50°C and 50 μl elution buffer 2 (TE, 1% SDS, 300 mM NaCl) for 15 min at 50°C while shaking. Both elutions were combined and incubated with proteinase K and RNase A for 2 hours at 37°C. DNA-protein complexes were de-crosslinked at 65°C while shaking overnight. ChIP DNA was purified using Agencourt AMPure XP beads (Beckman Coulter A63881).

[1] The bed.txt files generated from multiple samples are linked as Series supplementary files, described in the readme.txt in detail, and indicated in the corresponding sample description field.
[2] The bed.txt contains genomic coordinates of peaks and thus, can not be displayed in the genome browser.


title: lncRNA n384546 promotes thyroid papillary cancer proliferation and migration by targeting miR-145-5p to regulate AKT3

city: Shanghai

first: qinyi

last: zhou

address: Yishan Road 600

department: Department of Otolaryngology Head and Neck Surgery

institute: Shanghai Jiao Tong University Affiliated Sixth People's Hospital

summary: we document a novel lncRNA, n384546, which may exert its oncogenic property in PTC tumorigenesis by sponging miR-145-5p and then regulating its target AKT3.This study reveals that n384546 is an oncogenic lncRNA in human thyroid cancer.

overall_design: RNA-seq was performed in human papillary thyroid cancer tissue sample and adjacent normal tissue sample

first: Jiajia

last: Feng

first: Qinyi

last: Zhou


title: Increased expression of Kifc1 and Kifc5b targeting endogenous-siRNA contribute to the age-related decline in oocyte quality

city: Callaghan

first: Bettina

last: Mihalas

state: NSW

address: Level 4 Life Science Building, University Drive

department: Life Sciences

country: Australia

institute: University of Newcastle

summary: The oocyte is reliant on stores of endogenous mRNA transcripts in order to support their survival and integrity during a protracted period of dormancy as they await ovulation. Oocytes are however, known to experience an age-associated alteration in mRNA transcript abundance, a phenomenon that contributes to reduced developmental potential. Here we have investigated whether the expression profile of small non-coding RNAs (sncRNAs) is similarly altered in aged mouse oocytes. The application of high throughput sequencing revealed substantial changes in the global sncRNA profile of germinal vesicle stage oocytes from young (4-6 weeks) and aged mice (14-16 months). Among these changes, we identified 160 endo-siRNA and ten microRNAs that differentially accumulated within young and aged oocytes. Several endo-siRNAs that were upregulated in aged oocytes selectively targeted Kifc1 and Kifc5b; members of the kinesin protein family that were confirmed as being reciprocally reduced in the aged oocytes. The implications of reduced Kifc1 and Kifc5b expression were explored using complementary siRNA-mediated knockdown and pharmacological inhibition strategies, both of which led to increased rates of aneuploidy in otherwise healthy young oocytes. Collectively, our data raise the prospect that dysregulation of sncRNA expression, particularly endo-siRNA, may contribute to the age-dependent deterioration of oocyte quality.

overall_design: RNA-Seq was performed on a single biological replicate comprising of 582 young and 521 aged pooled oocytes, which were isolated from 18 and 49 animals, respectively

first: Nicole

middle: J

last: Camlin

first: Miguel

last: Xavier

first: Alexandra

last: Peters

first: Janet

last: Holt

first: Jessie

last: Sutherland

first: Eileen

last: McLaughlin

first: Andrew

last: Eamens

first: Brett

last: Nixon


title: Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma

first: Pratiti

last: Bandopadhayay

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary: we used gene expression profiling to determine gene expression changes in sensitive and drug tolerant medulloblastoma cells to reexposure to BET-bromodomain inhibitors

overall_design: Cells were treated with 1uM of DMSO or JQ1 and RNA-extracted at 24 hours

first: Rameen

last: Beroukhim


title: Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs [ChIP-seq]

first: Rudolf

last: Jaenisch

address: 455 Main Street

institute: Whitehead Institute for Biomedical Research

summary: Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and miR290 super-enhancers (SE) in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyl-transferases and transcription factor binding on one side, and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the miR290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.

overall_design: H3K27ac ChIP-Seq experiments were performed on sorted T+G+, T+G-, T-G+, and T-G- cells from the SOX2-SE-TG and MIR290-SE-TG mouse ESC lines

first: Yuelin

last: Song

first: George

last: Bell

first: Prat

last: Thiru


title: Parallel analysis of 5'-RNA ends in PSTVd-M inoculated tomato plants

city: Sherbrooke

first: Charith Raj

last: Adkar-Purushothama

state: Quebec

address: 3201 rue Jean-Mignault

department: Biochemistry

institute: Université de Sherbrooke

summary: 5′ RNA ligase mediated rapid amplification of cDNA ends (5′ RLM-RACE) is the widely used tool for confirming the direct cleavage of target mRNA by a miRNA. Recently, 5′ RLM-RACE combined with high-throughput sequencing such as parallel analysis of RNA ends (PARE) are gaining importance as these techniques allows to assess the large number of sample in a single experiment. In order to validate the cleavage of predicted a target mRNA in potato spindle tuber viroid-mild (PSTVd-M) infected tomato plants, the leaf samples collected at 21-days post inoculation were subjected for RNA extraction. A custom RNA adaptor containing MmeI restriction endonuclease was ligated to the free 5'-phosphate end of an uncapped mRNA using T4 RNA ligase, followed by reverse transcription (RT) and second strand synthesis using poly (T) primers. Obtained product was slightly amplified, cleaved with MmeI restriction endonuclease and then, a dsDNA adaptor was added to the 3’-end of the MmeI restriction endonuclease cleavage site. Resulted products were sequence in  an Illumina Mi-Seq machine. Retrieved data was then used to verify the 5’-termini of predicted target mRNA.

overall_design: Verification of RISC mediated cleavage of target mRNAs by PSTVd-M derived small RNA.

first: Charith

first: Jean-Pierre

last: Perreault


title: Transcriptional profiling of Ampk-deficient B cells during terminal differentiation

first: Fasih

middle: Mubtasim

last: Ahsan

address: 185 Cambridge Street, CPZN 6500

department: Center for Genomic Medicine

institute: Massachusetts General Hospital

summary: 5’-AMP-Activated Protein Kinase (Ampk) is an energy gatekeeper that responds to decreases in available ATP by inhibiting energy-consuming processes like protein synthesis and promoting energy-generating processes like glucose uptake. Recently, our lab showed that Lkb1, a previously unstudied kinase in B cell immunology and the main upstream kinase of Ampk, had a critical and unexpected role in B cell activation and germinal center formation. In T cells knockout of the downstream target Ampk, only partially phenocopies Lkb1 deletion, so we sought to determine whether the role of Lkb1 in B cells depends on Ampk. We find Ampk is activated upon B cell stimulation in vitro. Surprisingly, however, Ampk activation occurs in the absence of energetic stress, and B cells continue to grow and divide despite Ampk activity. We performed an IP-MS substrate screen to identify Ampk targets and only identified a limited repertoire of canonical targets, and many non-canonical targets. Despite activation of Ampk and the major role of Lkb1 in B cell activation, B cell specific deletion of Ampk does not significantly affect B cell activation, differentiation, carbon handling, gene expression, or humoral immune responses. The only major effect of Ampk loss was accelerated downregulation of IgD during stimulation, which was preceded by downregulation of the IgD regulator Zfp318. Early activation of Ampk by pharmacological means also has little impact on B cell function, although treatment with the biguanide Phenformin critically impairs germinal center formation and class switch recombination in vivo, but does not significantly impair antigen-specific antibody responses. Combined, our results suggest an unexpected and unexplained activation of Ampk in B cells.

overall_design: Bulk RNA-sequencing of CD43- sorted CD19-Cre+/-Prkaa1-/- (KO) and CD19-Cre+/-Prkaa1+/+ (WT) splenic naïve B cells (n = 3 each genotype), stimulated using CD40L + IL4 in vitro to terminal differentiation over 5 days (n = 3 each genotype at 5 timepoints plus naive, 36 samples total).

first: Lynnea

last: Waters

first: Michael

last: Teitell


title: ProMyelocytic Leukemia protein IV (PMLIV) represses breast cancer proliferation by modulating the FOXO3-FOXM1 axis

city: Heraklion

first: Joseph

last: Papamatheakis

state: Crete

address: Nikolaou Plastira 100

country: Greece

institute: Foundation for Research and Technology - Hellas

summary: The multitasking ProMyelocytic Leukemia (PML) protein, which was originally recognized as tumor suppressive factor, is commonly lost in primary breast cancer tissue samples. However, PML’s role in breast cancer is still debated, as high PML levels reportedly correlate with cancer cell pro-survival activity and poor patient prognosis in a subset of breast cancer types. To study the molecular mechanisms underlying the involvement of PML in cell proliferation and self-renewal pathways in breast cancer, a transgene expressing PMLIV upon doxycycline treatment was stably integrated in MDA-MB-231 cells (tetPMLIV MDA-MB-231 cells), a claudin-low, triple negative, aggressive breast cancer cell line.  Here, we report that inducible PMLIV expression leads to reduced proliferation rates and self-renewal of breast cancer cells as well as to impaired cell cycle progression. To further dissect PML’s role in breast cancer growth, we performed genome-wide expression analysis on MDA-MB-231 PMLIV overexpressing (OE) and control cells cultured in monolayer as well as in sphere conditions. Transcriptomic profiling identified many deregulated genes and their cognitive transcription factors. Upon PMLIV induction the Forkhead box subclass M1 (FOXM1) factor is itself suppressed at both the RNA and protein level, and consequently many of its cell cycle target genes. We show that FOXM1 interacts with PMLIV primarily via its DNA binding domain and that dynamically co-localizes in PML nuclear bodies. In parallel, PMLIV enhances the activity of FOXO3, a factor opposing some of the FOXM1 outputs, to promote cell survival and stress resistance. We propose that PMLIV may favour both anti-proliferative and pro-survival pathways at variable levels, dictated by genetic or epigenetic cancer cell states that may account for its disparate effects in cancer.

overall_design: We analyzed the gene expression profile of MDA-MB-231 culture in monolayer and sphere conditions before and upon PMLIV overexpression. PMLIV was induced for 48h.

first: N

last: Sachini

first: A

last: Klonizakis

first: C

last: Nikolaou

first: J


title: Consequences of a loss of Ash2l in the bone marrow.

city: Aachen

first: Bernd

last: Denecke

state: NRW

address: Pauwelsstrasse 30

department: IZKF Aachen

institute: RWTH Aachen University

summary:
The Ash2l protein is a member of KMT2 enzyme complexes, which catalyse the (tri-)methylation of lysine 4 of Histone H3. H3K4me3 is considered a marker of actively transcribed genes. We determined changes in gene expression as a consequence of a conditional loss of Ash2l in the bone marrow.
The expression data from the bone marrows of mice with floxed Ash2l exon 4 alleles without (control) or with an Mx1-Cre transgene (KO) and treated with synthetic dsRNA to induce recombination and the loss of Ash2l protein expression were compared.

overall_design: The expression data from LSK cells of the bone marrow from 3 control and 3 Ash2l KO mice were analysed and the fold changes calculated.

first: Juliane

last: Lüscher-Firzlaff

first: Nicolas

last: Chatain

last: Kuo

first: Ivan

last: Costa

first: Steffen

last: Koschmieder

first: Bernhard

last: Lüscher


title: Two miRNA signatures for identifying nasopharyngeal carcinoma from head-neck tumors and the normal

first: Mei-Yin

last: Zhang

state: Guangdong

address: 651 Dongfeng East Road

department: State Key Laboratory of Oncology in South China

summary: Reports show that miRNAs have been implicated in various cancers and functions as both oncogenes and tumor suppressors in tumour initiation, progression, and response to treatment. Our study is conducted to identify circulating miRNA profile in NPC patients using whole blood samples with clinical significant diagnostic value. Microarray profiling identified 117 differentially expressed miRNAs (fold change≥1.5) between NPC patients and healthy control and 164 differentially expressed miRNAs (fold change≥1.5) between NPC and HNT patients and healthy control. Among them, we obtain two miRNA signatures to help to diagnose NPC.

overall_design: All samples are randomly assigned to training group and validation group. By Lasso regression and adaptive boosting, we construct a diagnostic model in training set, and verify the accuracy of the microRNA signature in validation set.

first: Wen

last: Wen

first: Hui

last: Wang

first: Ling

last: Guo


title: Genome-wide expression from the esophageal biopsies of subjects with and without eosinophilic esophagitis

city: Cin

first: Leah

last: Kottyan

state: OH

address: 3333 Burnet Ave

summary: Esophageal biopsy RNA was isolated from distal esophageal biopsy RNA from EoE patients with active disease and from unaffected controls. EoE biopsies showed active disease pathology at the time when they were taken, and all patients reported no glucocorticoid treatment at the time of biopsy.   RNA sequencing acquiring 50 million mappable 125 base-pair reads from paired-end libraries was performed at the Genetic Variation and Gene Discovery Core Facility at Cincinnati Children's Hospital Medical Center. Data were aligned to the hg19 build of the human genome using the Ensembl annotations as a guide for TopHat.

overall_design: RNA sequencing of esophageal biopsy RNA


title: AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 5]

city: New York

first: Mariko

last: Kobayashi

state: NY

address: 1230 York Avenue

department: Molecular Neuro-Oncology

institute: Rockefeller University

summary: Cortical cultures treated with OGD (oxygen glucose deprivation) in the presence of miR-29 expressing lenti-virus were analyzed by RNAseq to assess differences in miR-29 target gene expression.

overall_design: DIV6 cortical cultures were infected with virus expressing miR-29 or empty control virus at (MOI=1) two times, for total MOI of 2.  On DIV14, cultures were first rinsed twice with warm OGD buffer (125 mM NaCl, 5 mM KCl, 1.2 mM Na2PO4, 26 mM NaHCO3, 1.8 mM CaCl2, 0.9 mM MgCl2, 10 mM Hepes, pH 7.4) plus 10 mM glucose and incubated for 30 min with the same rinsing buffer. The cultures were then rinsed with OGD buffer twice and transferred to an OGD chamber (Billups-Rothenberg, San Diego, CA) and flushed with anoxic gas (95% N2 and 5% CO2) at 21 L/min for 5 min. The chamber was then sealed and placed into 37°C incubator for 3 hrs. Controls (sham) were rinsed with OGD buffer but incubated in a normoxia conditions. After OGD treatment, the cultures were transferred to normal culture medium and incubated for 4 hrs, cells harvested and RNA extracted for library construction.  Each set of biological replicates represent cultures dissected on distinct days.  Complete protocol detailed in referenced publication.


title: AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 1]

summary: Adult mouse cortical tissues after MCAO or sham surgery were analyzed by Argonaute HITS-CLIP to assess differences in transcriptome-wide AGO binding patterns in I/R injury model of stroke.

overall_design: Mice were anesthetized briefly with 1.5–2% isoflurane.  A heat-blunted nylon suture (6/0) was inserted into the right external carotid artery and advanced until it obstructed the MCA together with the ligation of the common carotid artery for 35 min. Regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) was continuously recorded by transcranial laser Doppler flowmetry from the induction of ischemia until 30 min after reperfusion.  Surgery success was determined by monitoring regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) using transcranial laser Doppler flowmetry, and required a residual CBF <15%, followed by >80% recovery within 10 min of reperfusion.   Following MCAO, mice were placed in temperature-controlled recovery cages for 3 h to prevent post-surgery hypothermia and sacrificed.  Sham animals were similarly anestheized, arteries exposed for surgical time period without filament insertion.  Cortical tissues from ipsilateral or contralateral hemispheres of MCAO or sham animals were dissected, UV-irradiated and analyzed by CLIP protocol.  For each CLIP sample, high and low molecular weight AGO:RNA complexes (~110-130 kDa, Supplementary figure 1B) were pooled for library construction.  Each set of biological replicates were prepared separately by AGO-CLIP and consist of representative samples from a distinct day of surgery.  Complete protocol detailed in referenced publication.


title: AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 2]

summary: Adult mouse cortical tissues after MCAO or sham surgery were analyzed by RNAseq at various time points after reperfusion to assess differences in mRNA abundance in I/R injury model of stroke.

overall_design: Mice were anesthetized briefly with 1.5–2% isoflurane.  A heat-blunted nylon suture (6/0) was inserted into the right external carotid artery and advanced until it obstructed the MCA together with the ligation of the common carotid artery for 35 min.  Regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) was continuously recorded by transcranial laser Doppler flowmetry from the induction of ischemia until 30 min after reperfusion.  Surgery success was determined by monitoring regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) using transcranial laser Doppler flowmetry, and required a residual CBF <15%, followed by >80% recovery within 10 min of reperfusion.   Following MCAO, mice were placed in temperature-controlled recovery cages for 3, 6, 12 or 24 hrs to prevent post-surgery hypothermia and sacrificed.  Sham animals were similarly anestheized, arteries exposed for surgical time period without filament insertion.  Cortical tissues from ipsilateral or contralateral hemispheres of MCAO or sham animals were dissected for RNA extraction and library construction.  Each set of biological replicates represent animals from a distinct day of surgery.  Complete protocol detailed in referenced publication.


title: AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 4]

summary: Adult mouse cortical tissues after MCAO or sham surgery were analyzed by ribosomal profiling to assess differences in ribosome associated mRNA fragments in I/R injury model of stroke.

overall_design: Mice were anesthetized briefly with 1.5–2% isoflurane.  A heat-blunted nylon suture (6/0) was inserted into the right external carotid artery and advanced until it obstructed the MCA together with the ligation of the common carotid artery for 35 min.  Regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) was continuously recorded by transcranial laser Doppler flowmetry from the induction of ischemia until 30 min after reperfusion.  Surgery success was determined by monitoring regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) using transcranial laser Doppler flowmetry, and required a residual CBF <15%, followed by >80% recovery within 10 min of reperfusion.   Following MCAO, mice were placed in temperature-controlled recovery cages for 3, 6, 12 or 24 hrs to prevent post-surgery hypothermia and sacrificed.  Sham animals were similarly anestheized, arteries exposed for surgical time period without filament insertion.  Cortical tissues from ipsilateral or contralateral hemispheres of MCAO or sham animals were dissected for MNase digestion, monosome purificaton, large ribosomal subunit (P0) immunoprecipitaton, RPF (ribosome protected fragment) extraction and library construction.  Each set of biological replicates represent animals from a distinct day of surgery.  Complete protocol detailed in referenced publication.


title: Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog selective inhibitors for use in acute myeloid leukemia

first: Gabriela

last: Alexe

address: 415 Main St.

institute: Broad Institute

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

overall_design: Refer to individual Series


summary:
Exploiting an Asp-Glu “switch” in glycogen synthase kinase 3 to design paralog selective inhibitors for use in acute myeloid leukemia: Genome-wide transcriptional profiles for the GSK3α selective inhibitor BRD0507 and for the GSK3α/β dual inhibitor BRD0320
Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the WNT pathway, remains a therapeutic target of interest in many diseases. While dual GSK3α/β inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, are a concern in the translation of this target class to cancer therapy, particularly for the treatment of acute myeloid leukemia (AML). Knockdown of GSK3α or GSK3β individually does not increase β-catenin in certain cellular subtypes and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, only inadequate chemical tools exist.  The design of selective ATP competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity, 100% similarity) in their ATP binding domains. Taking advantage of an Asp133®Glu196 “switch” in their hinge binding domains, we present a rational design strategy towards the discovery of a paralog selective set of GSK3 inhibitors. These first-in-class GSK3α and GSK3β selective inhibitors provided insights into GSK3 targeting in AML where GSK3α has been identified as a therapeutic target using genetic approaches. Our GSK3α selective compound (BRD0705) inhibits kinase function and does not stabilize β-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells while no effect is observed on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies validate feasibility of paralog selective GSK3α inhibition offering a promising therapeutic approach in AML.

overall_design: Genome-wide transcriptional profiles for the GSK3α selective inhibitor  BRD0507 and for the GSK3α/β dual inhibitor BRD0320 in U937 cells.

first: Kimberly

last: Stegmaier