title: A Phase 2 Randomized Trial of Apremilast in Patients With Atopic Dermatitis

status: Public on Oct 06 2018

first: Ana

last: Pavel

address: One Gustave L. Levy Place

department: Dermatology

summary: A phase 2, double-blind, placebo-controlled trial evaluated apremilast efficacy, safety, and pharmacodynamics in adults with moderate to severe atopic dermatitis (AD).

overall_design: Patients were randomized to placebo, apremilast 30 mg BID (APR30), or apremilast 40 mg BID (APR40) for 12 weeks. During Weeks 12–24, all patients received APR30 or APR40. A biopsy substudy evaluated AD-related biomarkers. Among 185 randomized intent-to-treat patients at Week 12, a dose-response relationship was observed; APR40 (n=63), but not APR30 (n=58), led to statistically significant improvements (vs. placebo [n=64]) in Eczema Area and Severity Index (mean [SD] percentage change from baseline: −31.6% [44.6] vs. −11.0% [71.2]; P<0.04; primary endpoint). mRNA expression of Th17/Th22-related markers (IL-17A, IL-22, S100A7/A8; P<0.05) showed the highest reductions with APR40, with minimal changes in other immune axes. Safety with APR30 was largely consistent with apremilast’s known profile (common adverse events [AEs]: nausea, diarrhea, headache, nasopharyngitis). With APR40, AEs were more frequent and cellulitis occurred (n=6). An independent safety monitoring committee discontinued the APR40 dose. APR40 demonstrated modest efficacy and decreased AD-related biomarkers in moderate to severe AD patients. AEs, including cellulitis, were more frequent with APR40, which was discontinued during the trial.

first: Eric

last: Simpson

first: Shinichi

last: Imafuku

first: Yves

last: Poulin

first: Benjamin

last: Ungar

first: Lisa

last: Zhou

first: Kunal

last: Malik

first: Huei-Chi

last: Wen

first: Hui

last: Xu

first: Yeriel

middle: D

last: Estrada

first: Xiangyu

last: Peng

first: Mindy

last: Chen

first: Nilam

last: Shah

first: Mayte

last: Suarez-Farinas

first: Kristine

last: Nograles

first: Emma

last: Guttman-Yassky


title: SOX4 binding profiling in mouse embryonic neural stem cells (NSCs)

city: Utrecht

first: Paul

middle: J

last: Coffer

address: Uppsalalaan 6

department: Regenerative Medicine Center

country: Netherlands

institute: UMC Utrecht

type: Genome binding/occupancy profiling by high throughput sequencing

summary: By chromatin-immunoprecipitating (ChIP) SOX4 in NSCs we identified genome-wide binding sites for SOX4

overall_design: SOX4 ChIP-sequencing of mouse embryonic neural stem cells derived from E14.5 CD-1 mice

first: L

last: Braccioli

first: S

last: Vervoort

first: P


title: RNA-seq tumor immunome analysis of murine breast cancer model EMT6-hHER2 responsive to novel antibody drug conjugate T-PNU

city: Basel

first: Ulrike

last: Menzel

address: Mattenstrasse 26

department: Department of Biosystems Science and Engineering

country: Switzerland

institute: ETH Zürich

type: Expression profiling by high throughput sequencing

summary: An orthotopic murine breast cancer model was created by transfecting the human HER2 gene into the breast cancer cell line EMT6 resulting in EMT6-hHER2. Previous in vivo tumor treatments with trastuzumab, trastuzumab-emtansine (T-DM1) and a novel antibody drug conjugate (ADC) T-PNU (provided by NBE therapeutics and carrying the highly potent anthracycline analogue PNU-159682) revealed that EMT6-hHER2 tumors are unresponsive to standard therapy treatments of trastuzumab and T-DM1, but respond well to the novel ADC T-PNU. Identifying immunogenic cell death properties of T-PNU, we postulated a therapeutic relevance for T-PNU mediated immune modulation of the tumor microenvironment. In order to identify the transcriptional pathways underlying the T-PNU anti-tumor response, we performed RNA-sequencing analysis of CD45+ tumor-derived cells from differently treated cohorts (untreated, trastuzumab, T-DM1, T-PNU; n=6).

overall_design: mRNA of CD45+ tumor-immune cells from differently treated murine cohorts were sequenced by Illumina NextSeq.

first: Lucia

last: D'Amico

first: Michael

last: Prummer


title: Altered binding of tumor antigenic peptides to MHC class I affects CD8 T cell effector responses

city: Boston

first: Stephanie

last: Dougan

state: MA

address: 1 Jimmy Fund Way

department: Cancer Immunology & Virology

country: USA

institute: Dana-Farber Cancer Institute

summary: T cell priming occurs when a naïve T cell recognizes cognate peptide-MHC complexes on an activated antigen presenting cell. The circumstances of this initial priming have dramatic ramifications on the fate of the newly primed T cell. Newly primed CD8 T cells can embark onto different trajectories, with some becoming short lived effector cells and others adopting a tissue resident or memory cell fate. To determine whether T cell priming influences the quality of the effector T cell response to tumors, we used transnuclear CD8 T cells that recognize the melanoma antigen TRP1 using TRP1high or TRP1low TCRs that differ in both affinity and fine specificity. From a series of altered peptide ligands, we identified a point mutation (K8) in a non-anchor residue that, when analyzed crystallographically and biophysically, destabilizes the peptide interaction with the MHC binding groove. We then compared the transcriptional profiles of TRP1-specific T cells activated in vitro with M9 peptide to those activated with K8 peptide.

overall_design: Four samples were analyzed: TRP1high or TRP1low cells stimulated by B cells pulsed with K8 or M9 peptide for 48 hours.

first: Eleanor

last: Clancy-Thompson

first: Christine

middle: A

last: Devlin

middle: M

last: Tyler

first: Mariah

last: Servos

first: Lestat

middle: R

last: Ali

first: Katherine

middle: S

last: Ventre

first: M

last: Bhuiyan

first: Patrick

middle: T

last: Bruck

middle: E

last: Birnbaum

middle: K


title: The effects of IIS/TOR signaling on gene expression in Drosophila males and females: IIS/TOR impacts sex-differential gene expression

city: Auburn

first: Rita

middle: M.

last: Graze

state: AL

address: 101 ROUSE LIFE SCIENCES BLDG, AUBURN UNIVERSITY

department: Biological Sciences

institute: Auburn University

summary: The insulin signaling pathway is known to interact directly and indirectly with the sex determination regulatory hierarchy, playing a role in directing sexually dimorphic traits. To understand the degree to which the insulin signaling pathway contributes to sexually dimorphic gene expression in adult animals, we examined the effect of perturbation of the pathway on gene expression in male and female Drosophila. Our data reveal a large effect of insulin signaling on expression in adult males and females, with greater than 50% of genes examined changing expression. Males and females have a shared gene expression response to knock-down of InR function, with significant enrichment for pathways involved in metabolism. However, perturbation of insulin signaling has a greater impact on gene expression in males, with more genes changing expression and with a larger magnitude of changes overall. Primarily as a consequence of the response in males, we find that reduced insulin signaling results in a striking increase in sex-differential expression. This includes sex-differences in expression of immune, defense and stress response genes, as well as genes in the insulin signaling pathway itself. Our results demonstrate that perturbation of insulin signaling results in thousands of genes displaying sex differences in expression that are not differentially expressed in control conditions. Thus, insulin signaling may play a role in variability of sex-differential expression observed in studies of adult somatic tissues.

overall_design: Ubiquitous expression of a dominant-negative, insulin-like receptor transgene was induced using GeneSwitch in adult Drosophila virgin males and females and compared to age-matched controls. Two female samples (InR_EtOH_F2, InR_RU486_F1) were not analyzed due to low coverage, and are not included here.

first: Michelle

middle: N

last: Arbeitman


title: Transcriptomic Profile of OCI-AML-20 Cell Line

status: Public on Oct 05 2018

city: Toronto

first: Mark

last: Minden

state: Ontario

address: 610 University Avenue

department: Medical Oncology & Hematology

country: Canada

institute: Princess Margaret Cancer Centre

summary: Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by low response rate to induction type chemotherapy and hence is among the worst long term survivorship of the AMLs. Here, we present RNA-Seq transcriptome data from OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7.

overall_design: RNA-Seq transcriptome analysis of OCI-AML-20 cell line with three biological replicates.


title: Gene expression analysis of HP1B mutants in D. melanogaster.

city: Birmingham

first: Nicole

middle: Christina

last: Riddle

address: 1720 2nd Ave South CH464

department: Biology

institute: University of Alabama at Birmingham

summary: We profile gene expression changes in two mutant strains lacking the D. melanogaster HP1 homolog HP1B at the third instar larval stage. Compared to the yw control strain, several hundred genes are deregulated, with metabolic genes being over-represented among the deregulated gene set.

overall_design: Examination of gene expression in two genotypes

middle: C


title: Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced prostate cancer

city: Sacramento

first: Clifford

middle: G.

last: Tepper

state: CA

address: 4645 2nd Avenue, Room 2300A

department: Biochemistry and Molecular Medicine

institute: UC Davis School of Medicine

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

overall_design: Refer to individual Series


title: Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced prostate cancer (Affymetrix)

summary: Constitutively active androgen receptor (AR) signaling confers resistance to AR-targeted therapies. Here we show that the ubiquitin-mediated proteolysis pathway plays a critical role in the degradation of AR and its variants, particularly variant 7 (AR-V7). AR/AR-V7 proteinhomeostasis (proteostasis) requires interaction of the E3 ubiquitin ligase STUB1 and HSP70complex. STUB1 disassociates AR/AR-V7 from HSP70, leading to AR/AR-V7 ubiquitination and degradation. Inhibition of HSP70 reduces AR/AR-V7 expression which significantly inhibits prostate tumor growth and improves enzalutamide/abiraterone treatments. Clinically, HSP70 expression is upregulated and correlated with AR/AR-V7 levels in high Gleason scores prostate tumors. Our results reveal a novel mechanism of AR-V7 modulation via proteostasis which could be targeted to reduce AR-V7 expression and overcome resistance to AR-targeted therapies.

overall_design: Gene expression profiling of drug-resistant LNCaP-C42B castration-resistant prostate cancer (CRPC) cells was performed in order to define genes exhibiting altered expression relative to the untreated, LNCaP-C42B parental cell line. For this, drug-resistant cells were generated and maintained by long-term culture in enzalutamide (MDV3100) or abiraterone. Total cellular RNA was isolated and then microarray gene expression profiling performed with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays.

first: Chengfei

last: Liu

first: Allen

last: Gao

middle: G


title: Genome wide RNA-seq of wild-type and Safa-/- BMDMs upon HSV-1 infection.

city: Beijing

first: lili

last: Cao

address: Xueyuan road, haidian district

country: China

institute: Peking university health science center

summary: SAFA deficiency affects various gene expression upon HSV-1 infection.

overall_design: Wild-type and Safa-/- BMDMs (bone-marrow-derived macrophages cells) were infected with HSV-1 (herpes simplex virus type 1) for 6 hours. Genome wide RNA-Seq screen was performed to detect differential gene expression.

first: Lili

first: Fuping

last: You


title: MafK mediates chromatin remodeling to silence IRF8 expression in non-immune cells in a lineage-specific manner

city: Haifa

first: Nitsan

last: Fourier

address: Technion

country: Israel

institute: Technion

summary: The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors (TFs) and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific TF, and thus, serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. A high-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-seq analysis revealed three MafK binding-regions (-25kb, -20kb and IRF8 6th intron) in the IRF8 locus. These MafK binding-sites are sufficient to repress a reporter-gene when cloned in genome-integrated lentiviral reporter constructs in expression-restrictive cells only, while plasmid-based constructs do not demonstrate such repressive effect. Furthermore, removal of MafK-int6 binding-region from BAC-IRF8 reporter construct was sufficient to promote accessible chromatin conformation. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression only in non-immune cells.

overall_design: MafK ChIP-seq was performed in two cell lines, mouse fibroblast cell-line NIH3T3 and mouse macrophage cell-line RAW. For NIH3T3 cells, 4 IP replicates were analyzed and 1 input sample. For RAW cells 3 IP replicates were analyzed and 1 input sample.

first: Ben-Zion

last: Levi


title: Adult Connective Tissue-Resident Mast Cells Originate from Late Erythro-Myeloid Progenitors [origins]

city: Hangzhou

first: ZHI

middle: QING

last: LI

address: 866 Yuhangtang Road

institute: Zhejiang University School of Medicine

summary:
Purpose: The purpose is to systemically identify RNA expression between skin mast cells with distinct origins.
Methods: We profiled RNA-seq in skin mast cells with distinct origins.
Results: Early, late EMP and fetal HSC-derived skin mast cells display distinct transcriptome profiles.

overall_design: Skin mast cells were sorted from Csf1rCre/EYFP mice or Runx1Cre/EYFP mice at 1 week postnatal and then subjected to RNA-seq.

first: Xuetao

first: Zhiqing

last: Li


title: Defining T cell states associated with response to checkpoint immunotherapy in melanoma

city: Cambridge

first: Keren

last: Yizhak

address: 415 Main street

institute: Broad Institute of MIT and Harvard

summary: we profiled 16,291 immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors, using single-cell transcriptomics.

overall_design:
Tumors were disaggregated, sorted into CD45+ single cells, and profiled by Smart-seq2
-----------------------------------------------------
Submitter states "As this is human data, raw sequencing files are submitted via dbGap."

dbGaP accession code: phs001680.v1.p1

first: Moshe

last: Sade-Feldman


title: Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced prostate cancer (RNA-Seq)

overall_design: A total of 6 samples were analyzed in this study. The study included 2 different castration-resisatnt prostate cancer (CRPC) cell lines, LNCaP-C42B-MDVR and CWR22Rv1. Individual cultures of each cell line were treated with DMSO vehicle control, apoptozole, or VER155008. Following treatment of the cells for 48 hours, total RNA was isolated and then submitted for RNA-sequencing analysis.


title: Sm eggs Virtual memory T cells

city: Liege

first: Benjamin

last: Dewals

address: Avenue de Cureghem, 10

country: Belgium

institute: University of liege

summary: Transcriptomics analysis of virtual memory CD8+ T cells after treatment with Schistosoma mansoni eggs.

overall_design: RNA sequencing was peformed from virtual memory T cells obtained from the spleens of two groups of 3 BALB/c mice either treated with PBS (control) or treated with Schistosoma mansoni eggs.

first: Marion

last: Rolot

first: Annette

last: Dougall


title: Dissecting cell-intrinsic roles of MyD88 and IFN-I signalling in pDC responses to a viral infection in vivo

city: marseille

first: Thien-Phong

last: Vu Manh

address: 163 avenue de Luminy

country: France

institute: CIML

summary: Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN-I) during viral infections, in response to triggering of endosomal Toll Like Receptors (TLR) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. IFN-I production in pDC occurs in specialized endosomes encompassing preformed signaling complexes of TLR7 or 9 with their adaptor molecule MyD88 and the transcription factor interferon regulatory factor 7 (IRF7). The triggering of TLR leads to IRF7 phosphorylation, nuclear translocation and binding to the promoters of the genes encoding IFN-I to initiate their transcription. pDC express uniquely high levels of IRF7 at steady state and this expression is further enhanced by positive IFN-I feedback signaling during viral infections. However, the specific cell-intrinsic roles of MyD88 versus IFN-I signaling in pDC responses to a viral infection have not been rigorously dissected. To achieve this aim, we generated mixed bone marrow chimera mice (MBMC) allowing to rigorously compare the gene expression profiles of WT versus Ifnar1-KO or MyD88-KO pDC isolated from the same animals at steady state or after infection with the mouse cytomegalovirus (MCMV). Our results indicate that, in vivo during MCMV infection, pDC undergo a major transcriptional reprogramming, under combined instruction of IFN-I, IFN-γ and direct TLR triggering. However, these different stimuli drive specific, largely distinct, gene expression programs. We rigorously determined which gene modules require cell-intrinsic IFN-I signaling for their induction in pDC during a physiological viral infection in vivo. We delineated non-redundant versus shared versus antagonistic responses with IFN-γ. We demonstrated that cell-intrinsic IFN-I responsiveness is dispensable for induction of the expression of all IFN-I/III genes and many cytokines or chemokines in pDC during MCMV infection, contrary to MyD88 signaling.

overall_design: Recipient CD45.1 mice were lethally irradiated and reconstituted with a 1:1 mixture of BM cells from WT CD45.1 and Ifnar1-KO or MyD88-KO CD45.2 donor animals, to generate test mice (TST MBMC). Congenic CD45.1+ mice differ from the CD45.2+ C57BL/6 strain for several gene polymorphisms and immune parameters (Waterstrat et al., 2010). Therefore, as controls, we included mice engrafted with a mixture of WT CD45.1 and WT CD45.2 BM cells (CTR MBMC). MBMC were infected with MCMV at least 8 weeks after BM graft. Their splenic pDC were isolated 1.5 days after infection, at the peak of IFN-I production, sorted into CD45.1+ versus CD45.2+ populations, and used for pangenomic gene expression profiling by microarrays. Cells from regular, uninfected or d1.5 MCMV-infected, C57BL/6 mice were added as controls to ensure lack of major impact on pDC responses to in vivo MCMV infection of the bone marrow graft.

first: Elena

last: Tomasello

first: Anissa

last: Fries

first: Rabie

last: Chelbi

first: Gilles

last: Bessou

first: Emeline

last: Pollet

first: Thien

middle: P

first: Marc

last: Dalod


title: High-resolution, in vivo genome binding interactions of the mouse and human constitutive androstane receptors in rodent liver reveal species differences and novel cancer-related target genes

status: Public on Oct 08 2018

city: University Park

first: Curtis

middle: J.

last: Omiecinski

state: PA

address: 101 Life Sciences Bldg

department: Veterinary & Biomedical Science

institute: Penn State University

summary: The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor orchestrating xenobiotic drug metabolism and endobiotic energy homeostasis. Emerging roles for species differences in CAR’s regulation of hepatocyte proliferation and as an effector of hepatocarcinogenesis remain poorly understood, although several lines of evidence support the concept that mouse CAR and human CAR differentially program these processes. In this investigation, we conducted whole genome mapping of CAR-DNA interactions using high resolution ChIP-exo protocols combined with an adenovirus delivery strategy allowing transient expression of the respective receptors in livers of transgenic mice. The results obtained were informative in several respects, revealing for example CAR binding to novel target genes, including Gdf15 and Foxo3, previously characterized as regulators of carcinogenic process, and identification of species differences in the genomic interactions of mouse and human receptors that program altered expression profiles for the proto-oncogenes, Myc and Bmf. The ChIP-exo analyses also allowed for characterization of high-resolution mCAR and hCAR binding motifs across the genome. Together, the results obtained help clarify CAR’s role as a regulator of biological signaling processes involved in cell proliferation and tumorigenesis, and provide new insights into species variation in liver tumor promotion.

overall_design: Adenovirus YFP-hCAR and YFP-mCAR constructs were delivered to CAR (-/-) knockout mice, total liver was harvested for ChIP-exo to investigate binding regions of hCAR and mCAR in C57/Bl6 mice. Following delivery, mice were treated by species specific CAR direct activators (1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; TCPOBOP, for mouse; 6-(4-chlorophenyl: imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; CITCO, for human), or the indirect activator, phenobarbital. 3 biological replicates for each treatment. Total 12 samples. Adenovirus YFP-empty constructs (n=2) were used as controls for ChIP-exo analysis.

first: Ben

last: Niu

first: Denise

last: Coslo

first: Alain

last: Bataille

first: Istvan

last: Albert

first: B

middle: F

last: Pugh


title: Transcription-associated histone pruning demarcates macroH2A chromatin domains

city: New York

first: Emily

last: Bernstein

state: NY

address: 1470 Madison Avenue, 6th floor Rm 302

department: Oncological Sciences

institute: Mount Sinai School of Medicine

summary: Here, we characterized de novo chromatin deposition of macroH2A2 using temporal genomic profiling in cells devoid of all macroH2A isoforms. We find that macroH2A2 is first pervasively deposited genome-wide and subsequently pruned to establish mature domains. This transient incorporation occurs preferentially at transcribed regions adjacent to future mature macroH2A2 domains and transcriptional inhibition prevents the clearing of promiscuously incorporated macroH2A2. Furthermore, CRISPR/Cas9-based manipulation of the transcriptional activity at a given locus reveals that transcriptional silencing triggers ectopic macroH2A2 accumulation, while activation depletes pre-existing macroH2A2. We demonstrate that the FACT (facilitates chromatin transcription) complex is required for macroH2A2 removal from transcribed chromatin. Taken together, we have identified a transcription-associated pruning mechanism that establishes and maintains the faithful genomic localization of macroH2A variants.

overall_design: Native ChIP-seq (nChIP-seq) for macroH2A2-GFP was performed at 0, 6, 24 hours and 5 days after dox addtion in macroH2A1/2 dKO iDFs with inducible macroH2A2-GFP transgene. Additionally, nChIP-seq for macroH2A2-GFP was done 24 hours post dox induction with the last 18 hours under flavopiridol (CDK9 inhibitor) treatment. Further, inducible iDFs were treated with Ezh2 inhibitor GSK126 and native ChIP-seq for macroH2A2-GFP was performed 6 and 24 hours post dox addition. Input sample was sequenced as control. PolyA mRNA-seq was performed at 0, 6 and 24 hours after dox induction in biological triplicates in the same cell line.

first: Zhen

last: Sun


title: Longitudinal Personal DNA Methylome Dynamics in a Human with a Chronic Condition

city: Stanford

first: Michael

last: Snyder

address: 300 Pasteur Drive

department: Department of Genetics

institute: Stanford University

type: Methylation profiling by high throughput sequencing

summary: Epigenomics regulates gene expression and is as important as genomics in precision personal health, as it is heavily influenced by environment and lifestyle. We profiled whole genome DNA methylation and corresponding transcriptome of peripheral blood mononuclear cells collected from a human volunteer over a period of 36 months, generating 28 methylome and 57 transcriptome datasets. We found that DNA methylomic changes are associated with infrequent glucose levels alteration, whereas the transcriptome underwent dynamic changes during events such as viral infections. Most DNA meta-methylome changes occurred 80-90 days prior to clinically detectable glucose elevation. Analysis of the deep personal methylome dataset revealed an unprecedented number of allelic differentially methylated regions (aDMR) which remain stable longitudinally and are preferentially associated with allele-specific gene regulation. Our results revealed that different types of "omics" data associate with different aspects of conditions of this individual: DNA methylation with chronic disease and transcriptome with acute events.

overall_design: Whole genome bisulfite sequencing and RNA-Sequencing.

first: Rui

last: Chen

first: Dan

last: Xie


title: TET2 coactivates gene expression through demethylation of enhancers

status: Public on Oct 04 2018

city: Chicago

first: Ali

last: Shilatifard

state: IL

address: 320 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

summary: The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of ERalpha. Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA-methylation at enhancers resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERalpha, which further requires MLL3/COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

overall_design: To examine changes in histone modifications, ER-alpha occupancy, MLL3 occupancy, and gene expression changes after depletion of TET2 in human breast cancer cell lines.

first: Lu

last: Wang

first: Patrick

middle: A

last: Ozark


title: RNA-Seq analysis of human lens epithelial cells exposed to ionizing radiation

city: Ottawa

first: Matthew

last: Meier

address: 251 Sir Frederick Banting Driveway

department: Health Canada

institute: Government of Canada

summary:
Purpose: The International Commission on Radiological Protection (ICRP) recently recommended reducing the occupational equivalent dose limit for the lens of the eye.  Based primarily on a review of epidemiological data, the absorbed dose threshold is now considered to be 0.5 Gy for induction of acute opacities, reduced from the previous threshold of 2 Gy.    However, direct mechanistic evidence to support an understanding of the underlying molecular mechanisms of damage is still lacking. To this end, we explored the effects of a broad dose-range of ionizing radiation exposure on gene expression changes in a human lens epithelial (HLE) cell-line in order to better understand the shape of the dose-response relationship and identify transcriptional thresholds of effects.
Methods: HLE cells were exposed to doses of 0, 0.01, 0.05, 0.25, 0.5, 2, and 5 Gy of X-ray radiation at two dose rates (1.62 cGy/min and 38.2 cGy/min).  Cell culture lysates were collected 20 h post-exposure and analysed using whole-genome RNA-sequencing.  Pathways and dose-thresholds of biological effects were identified using benchmark dose (BMD) modeling.
Results: Transcriptional responses were minimal at doses less than 2 Gy. At higher doses there were a significant number of differentially expressed genes (DEGs) (p < 0.05, fold change > |1.5|) at both dose rates, with 1308 DEGs for the low dose rate (LDR) and 840 DEGs for the high dose rate (HDR) exposure. Dose-response modeling showed that a number of genes exhibited non-linear bi-phasic responses, which was verified by digital droplet PCR.    BMD analysis showed the majority of the pathways responded at BMD median values in the dose range of 1.5-2.5 Gy, with the lowest BMD median value being 0.6 Gy for the HDR exposure. The minimum pathway BMD median value for LDR exposure, however, was 2.5 Gy. Although the LDR and HDR exposures shared pathways involved in extracellular matrix reorganization and collagen production with BMD median value of 2.9 Gy, HDR exposures were more effective in activating pathways associated with DNA damage response, apoptosis, and cell cycling relative to LDR exposure.
Conclusion: Overall, the results suggest that radiation induces complex non-linear transcriptional dose-response relationships that are dose-rate dependent. Pathways shared between the two dose rates may be important contributors to radiation-induced cataractogenesis. BMD analysis suggests that the majority of pathways are activated above 0.6 Gy, which supports current ICRP identified dose thresholds for deterministic effects to the lens of the eye of 0.5 Gy.

first: Vinita

last: Chauhan

first: Andrea

last: Rowan-Carroll

first: Remi

last: Gagne

first: Byron

last: Kuo

first: Andrew

last: Williams

first: Carole

middle: L

last: Yauk


title: Glucocorticoid induced gene signature in human skin

first: Loukia

last: Lili

address: 770 Lexington Ave, 14-07 14th floor

department: Icahn School of Medicine at Mount Sinai

institute: The Institute for Next Generation Healthcare

summary: Glucocorticoids (GCs) are among the most effective and frequently prescribed anti-inflammatory drugs in dermatology. The effects of GCs are mediated by the glucocorticoid receptor (GR), a well-known transcription factor (TF). Despite the well-studied therapeutic and adverse effects of GCs, the molecular signature of GC/GR in human skin is largely unknown. Here, we present a bioinformatics analysis of the human skin transcriptome induced by the glucocorticoid clobetasol propionate (CBP). Full thickness arm skin biopsies were obtained from a cohort of 17 healthy volunteers balanced in sex and race (African-American and Caucasian) 24 hrs after single CBP topical application. Non-treated skin from the same volunteers served as control. Analyses of differentially expressed genes (DEGs) and gene set enrichments (GSE) from RNASeq reflected anti-inflammatory, metabolic and atrophogenic effects of GCs in skin.  More than 40% of all DEGs were non-coding RNAs, including long non-coding RNAs (lncRNAs) suggesting that GR effects in skin may extend towards regulation at the post-transcriptional level. Furthermore, we observed sexual and racial dimorphism in the CBP response including a shift towards IFNg/IFNa and IL6/JAK/STAT3 signaling in female skin; and an overall larger response to CBP (increased number of DEGs and increased induction of some GR-target genes) in African-American skin. Weighted gene co-expression network analysis (WGCNA) unveiled a regulatory network comprising 41 TFs, with network hub TFs enriched for direct GR targets and  ~ 260 of their target genes that were enriched for the majority of altered functional pathways of the entire CBP transcriptome. Interestingly, ~ 80% of these TFs involved in the regulation of circadian clock, metabolism, and development have been associated with skin aging supporting at the molecular level the known similarities between skin chronically treated with GCs and  aged skin. Overall, these findings indicate that GR acts as a master regulator of gene expression in skin via multiple mechanisms including regulation of expression of noncoding RNAs and multiple core TFs

overall_design: RNASeq data analysis of skin biopsies from 17 healthy volunteers

first: Anna

last: Klopot

first: Ben

last: Readhead

first: Gleb

last: Baida

first: Joel

last: Dudley

first: Irina

last: Budunova


title: CRISPR activation of long non-coding RNAs transiently expressed during cortical neuron differentiation associated with Field, et al, Stem Cell Reports 2018

middle: Ryan

last: Field

address: 45 Shattuck St

department: BCMP

institute: Harvard Medical School

summary: 4 transiently expressed long non-coding RNAs that were identified in human and non-human primate cortical organoid differentiation were activated out of context in HEK293FT cells using CRISPRa.

overall_design: 5 sgRNAs targeting TrEx lncRNAs or non-targeting controls were co-transfected with dCas9-VP64 into HEK293FT cells. Successfully transfected cells were selected by puromycin at 24 hours and harvested for RNA at maximal expression, 48 hours post transfection. RNA-seq libraries were prepared in biological triplicates with the NEXTflex Rapid Directional qRNA-Seq Library Prep Kit (PerkinElmer).

middle: R

first: Frank

middle: M

last: Jacobs

first: Ian

last: Fiddes

first: Alex

last: Phillips

last: Reyes-Ortiz

first: Erin

last: LaMontagne

first: Lila

last: Whitehead

first: Vincent

last: Meng

first: Jimi

last: Rosenkrantz

first: Mari

last: Olsen

first: Max

last: Hauessler

first: Sol

last: Katzman

first: Sofie

last: Salama

first: David

last: Haussler


title: The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination

city: NEW YORK

first: Thiago

middle: Y

last: Oliveira

state: New York

address: 1230 YORK AVE

department: Immunology, Virology and Microbiology

institute: The Rockefeller University

summary: Class Switch Recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3’ Igh super-enhancer, 3’ Regulatory Region (3’RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here we identify the chromatin reader ZMYND8 as an essential regulator of the 3’RR and CSR. In B cells, ZMYND8 binds promoters and super-enhancers, including the 3’RR, and controls its activity by modulating the enhancer transcriptional status. In its absence, there is increased 3’RR polymerase loading, and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3’RR. Thus ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3’ Igh super-enhancer.

overall_design: ChIP-seq, RNA-seq and GRO-Seq of wildtype or ZMYND8 KO CH12 or primary B cells.

first: Verónica

last: Delgado-Benito

first: Daniel

middle: B

last: Rosen

first: Qiao

last: Wang

first: Anna

last: Gazumyan

first: Joy

last: Pai

first: Devakumar

last: Sundaravinayagam

first: Wenzhu

last: Zhang

first: Matteo

last: Andreani

first: Lisa

last: Keller

first: Kyong-Rim

last: Kieffer-Kwon

first: Aleksandra

last: Pekowska

first: Seolkyoung

last: Jung

first: Madlen

last: Driesner

first: Roman

middle: I

last: Subbotin

first: Rafael

last: Casellas

first: Brian

middle: T

last: Chait

first: Michel

last: Nussenzweig

first: Michela

last: Di Virgilio


title: DNA methylation changes in Dnmt3L-deficient mouse embryonic stem cells (mESCs)

city: Houston

first: Yue

last: Lu

state: TX

address: 1881 East Road

department: Epigenetics & Molecular Carcinogenesis

institute: The University of Texas MD Anderson Cancer Center

summary: We used RRBS to analyze DNA methylation in mESC lines deficient for maternal Dnmt3L (Dnmt3L mKO), zygotic Dnmt3L (Dnmt3L KO), and both maternal and zygotic Dnmt3L (Dnmt3L mzKO). Compared to wild-type (WT) mESCs, Dnmt3L mKO mESCs exhibit severe loss of methylation at imprinted loci but no changes in global DNA methylation, Dnmt3L KO mESCs exhibit moderate loss of methylation at many Dnmt3a target regions but do not affect methylation at imprinted loci, and Dnmt3L mzKO mESCs exhibit combined changes of mKO and KO cells, with severe loss of methylation at imprinted loci and moderate loss of methylation at Dnmt3a target regions.

overall_design: Dnmt3L mKO, KO and mzKO mESC lines, as well as control WT mESC lines, were derived from blastocyst-stage embryos from intercrosses between Dnmt3L-/- or Dnmt3L+/- female mice and Dnmt3L+/- male mice. 2-3 male (XY) lines per genotype were analyzed by RRBS.

first: Taiping

last: Chen

first: Hongbo

last: Zhao


title: Genome-wide fork-collapse sites in MEFs from ATR inhibition [mouse]

city: Philadelphia

first: Nishita

last: Shastri

state: Pennsylvania

address: 421 Curie Boulevard

department: Cancer Biology

institute: University of Pennsylvania

summary: Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use a developed DNA break-detection assay, BrITL, to identify replication-problematic loci (RPLs) that become processed into persistent double-strand breaks across the mammalian genome from ATR inhibition.

overall_design: Examination of double-strand break sites in mouse embryonic fibroblasts arising from low-dose aphidicolin treatment and ATR inhibition. Two replicates of each condition are included, in addition to one input per condition, totaling 6 samples. This includes DMSO-treated controls. Samples were deep-sequenced through Illumina HiSeq.

middle: K

first: Eric

middle: J

last: Brown


title: Migration through a small pore disrupts inactive chromatin organisation in neutrophil-like cells [RNA-seq]

city: Auckland

first: Justin

middle: M.

last: O'Sullivan

address: 85 Park Road, Grafton

department: Liggins Institute

country: New Zealand

institute: University of Auckland

summary:
Background. Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. Their nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through constricted space, where the nuclear shape must change in order to fit through. This happens many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration.

Results. Total RNA-sequencing identified that neutrophil migration through 5 or 14 µm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes, when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeleton remodelling. Hi-C was used to capture the genome organisation in control and migrated cells. Chromatin did not switch between the active (A) and inactive (B) compartments after migration. However, global depletion of mid- to long-range contacts was observed following active migration through the 5 µm pores. Chromatin contacts that decreased in frequency were enriched in inactive chromatin.

Conclusion. Mid- to long-range contacts are preferentially lost within inactive chromatin, thus protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in human health and disease.

overall_design: Total RNA sequencing in triplicate of neutrophil-like cells migrated through large (14µm) or small (5µm) pores, with an unmigrated control population.

first: Elsie

middle: C

last: Jacobson


title: sc-RNA sequencing of skeletal muscle macrophages during T. gondii infection and injury

city: Buffalo

first: Elizabeth

last: Wohlfert

address: 955 Main Street

institute: University at Buffalo

summary: The proper coordination of macrophages is essential for skeletal muscle repair. However, the full heterogenity of macrophages responding to injury has yet to be elucidated on the single-cell level. Furthermore, chronic inflammation may shift macrophage heterogeneity at steady-state and affect the generation of heterogeneity necessary for tissue repair. We use scRNA-sequencing to determine the heterogeneity of skeletal muscle macrophages during infection and wound repair (uninfected and infected). We identify multiple transcriptionally distinct macrophage subsets during injury in uninfected mice as well as during infection at steady-state. Interestingly, in response to injury, infected skeletal muscle macrophages fail to generate certain subsets of reparative macrophages in a timely fashion.

overall_design: 8-12 week old C57BL/6 mice were orally infected with 5 ME49 T.gondii cysts and allowed to reach d24 post-infection to establish chronicity. Age and sex-matched uninfected and infected mice were given cardiotoxin naja pallida i.m. in one leg at 24 days post-infection. Contralateral leg was used as an uninjured control. Muscles were harvested at 4 days-post injury and prepared into a single cell suspension. Single cell suspensions were bulk sorted for macrophages by 7AAD-CD45+CD64+MerTK+ cells. Sorted cells from each group was individually assessed for and loaded onto the 10X Genomics Chromium platform for single-cell capture. cDNA libraries were constructed and subsequently sequenced on the Illumina platform. Experimental groups were performed in duplicate.

first: Richard

last: Jin


title: Genome-wide fork-collapse sites in MDA-MB-231 cells from ATR inhibition [human]

summary: Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use a developed DNA break-detection assay, BrITL, to identify replication-problematic loci that become processed into persistent double-strand breaks across the human genome from ATR inhibition.

overall_design: Examination of double-strand break sites in human triple-negative breast cancer cells arising from low-dose aphidicolin treatment and ATR inhibition. Two replicates of each condition are included, in addition to one input per condition, totaling 6 samples. This includes DMSO-treated controls. Samples were deep-sequenced through Illumina HiSeq.


title: RNA-sequencing of mouse thymic leukemias extracted from Mb1-CreDPB mice

city: London

first: Rodney

middle: Peter

last: DeKoter

address: 1151 Richmond St

department: Microbiology and Immunology

institute: Western University

summary: We examined the patterns of gene expression of mouse thymic leukemias extracted from Mb1-CreDPB mice by RNA sequencing (RNA-seq). Our goal was to integrate RNA-seq data with whole-exome sequencing (WES) to determined secondary driver mutations of leukemogenesis in the absence of Spi-B and PU.1,

overall_design: Thymic leukemias were isolated from diseased Mb1-CreDPB mice. In summary, thymuses were homogenized and red blood cells were removed with ACK buffer, washed with PBS  and counted. The amount of 8 million cells were pelleted an RNA was extracted using Rneasy RNA Isolation Kit (Qiagen). RNA was quantified and  the purity was checked by spectophotrometer.  RNA was sent to subsequently sequencing procedures.

first: Carolina

last: Batista


title: Optimal settings of mass spectrometry open search strategy for higher confidence

city: Guangzhou

first: Li

last: Dehua

state: Guangdong

address: Huangpu avenue west NO.601

institute: Jinan University

summary: More than half of the mass spectra could not be identified in most proteome mass spectrometry experiments. Various modifications have been considered as a major reason. Open search strategy was then introduced to solve this problem, however, lacking thorough quality assessment using independent information. Here, we used the “Suspicious Discovery Rate (SDR)” based on the translatome sequencing (RNC-seq) as an independent source to assess the proteome open search strategy. We found that the open search strategy increased the spectra utilization with the cost of increasing suspicious identifications that lacks translation evidence. We further suggested that restricting the peptide FDR below 0.1% would efficiently control the suspicious identifications of open search methods and enhanced the confidence of the peptide identification with modifications than the narrow window search. These results facilitated the proper use of open search methods for higher quality of proteome identifications with the information of post-translational modifications and single amino acid polymorphisms

overall_design: Getting translatome data of hela cell line and verifing some single nucleotide polymorphism sites in MHCC97H cell line

first: Shaohua

first: Zhibiao

last: Mai


title: Genetic analysis of isoform usage in the human anti-viral response

first: Jenny

address: 16 Divinity Ave

institute: Harvard

summary: While genetic variants are known to be associated with overall gene abundance in stimulated immune cells, less is known about their effects on alternative isoform usage. Analyzing RNA-seq profiles of monocyte-derived dendritic cells from 243 individuals, we uncovered thousands of unannotated isoforms synthesized in response to influenza infection and type 1 interferon stimulation. We identified more than a thousand quantitative trait loci (QTLs) associated with alternate isoform usage (isoQTLs), many of which are independent of expression QTLs for the same gene. Compared to eQTLs, isoQTLs are enriched for splice sites and untranslated regions, but depleted of sequences upstream of annotated transcription start sites. Both eQTLs and isoQTLs explain a significant proportion of the disease heritability attributed to common genetic variants.

overall_design:
We used paired-end RNA-seq to profile the transcriptomes of primary MoDCs from healthy donors at rest (N = 99), and following stimulation with either recombinant interferon beta (IFNB1), a type 1 interferon that stimulates anti-viral effectors (N = 227), or influenza ΔNS1 (a strain engineered to maximize the type 1 interferon-induced response to infection by the deletion of a key virulence factor).

Submitter declares that the raw data have been submitted to dbGaP (phs000815.v1.p1) because of patient privacy concerns.

first: Chun

last: Ye

first: Alexandra-Chloé

last: Villani

first: Meena

last: Subramaniam

first: Rachel

middle: E

last: Gate

first: Tushar

last: Bhangale

first: Mark

middle: N

last: Lee

first: Towfique

last: Raj

first: Raktima

last: Raychowdhury

first: Weibo

last: Li

first: Noga

last: Rogel

first: Sean

last: Simmons

first: Selina

middle: H

last: Imboywa

first: Portia

last: Chipendo

first: Cristin

last: McCabe

first: Michelle

first: Irene

last: Frohlich

first: Barbara

last: Stranger

first: Philip

last: De Jager

first: Aviv

last: Regev

first: Tim

last: Behrens

first: Nir

last: Hacohen


title: Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics - human

status: Public on Oct 03 2018

city: Chippendale

first: Darya

middle: Pavlovna

last: Vanichkina

state: New South Wales

address: 32 Queen Street

country: Australia

institute: University of Sydney

summary: MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3cyanovinylcarbazole ( CNV K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNV K-containing miR-29b ( CNV K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, arehighly sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNV K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and findrobust conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNV K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNV K-containing small RNA mimics may find applicability in other experimental models

overall_design: pulldown assay with 3x3x3x3 design: miRNA mimic and scrambled miRNA mimic x input and pulldown conditions

first: Anupma

last: Choudhary


title: Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: HepG2 Hi-C

city: Saarbrücken

first: Abdulrahman

last: Salhab

address: campus Saarbrücken

department: Genetics

country: Germany

institute: Saarland university

summary: Partially methylated domains, PMDs, are extended regions in the genome exhibiting a reduced average DNA-methylation level. PMDs cover gene-poor and transcriptionally inactive regions and tend to be heterochromatic. Here, we present a first comprehensive comparative analysis of PMDs across 171 and 24 WGBS methylomes of human and mouse cells, respectively, providing a deep insight into structural and functional features associated with PMDs.

overall_design:
Integrative analysis of partially methylated domains across 171 human and 24 mouse WGBS data sets
See samples_IDs.xlsx for details on the 171 human and 24 mice datasets

first: Jörn

last: Walter

first: Thomas

last: Manke


title: mRNA-seq for etoposide-treated TLP-knockdown cells

city: Yokohama

first: Hidefumi

last: Suzuki

address: Fukuura 3-9

country: Japan

institute: Yokohama City University

summary: We explored the mechanism by which TLP affects DNA damage response. We performed mRNA-seq analysis of non-treated and etoposide-treated samples in control and TLP-knockdown cells. We show that TLP is critical for global transcriptional repression after double-strand DNA breaks (DSBs).

overall_design: Examination of non-treated and etoposide-treated samples in control and TLP-knockdown cells

first: Yuki

last: Yamaguchi


title: Integrative epigenetic taxonomy of primary prostate cancer [ChIP-Seq]

city: Nijmegen

first: Suzan

last: Stelloo

address: Geert Grooteplein Zuid 28

department: Molecular Biology

institute: Radboud University

summary: The Androgen Receptor (AR) is the key-driving transcription factor in prostate cancer, tightly controlled by epigenetic regulation. To date, most epigenetic profiling has been performed in cell lines or limited tissue samples. To comprehensively study the epigenetic landscape, we complemented RNA-seq with ChIP-seq for AR and histone modification marks (H3K27ac, H3K4me3, H3K27me3) in 100 primary prostate carcinomas. Integrative molecular subtyping of the five data streams revealed three major subtypes of which two were clearly TMPRSS2-ERG dictated. Importantly, a third novel subtype was identified, with low AR chromatin binding and activity, even though the receptor was clearly expressed. While positive for neuroendocrine-hallmark genes, these tumors were copy number-neutral with low mutation burden, significantly depleted for genes characteristic of poor-outcome associated luminal B-subtype. We present a rich novel resource on transcriptional and epigenetic control in prostate cancer, revealing a tight control of gene regulation differentially dictated by AR over the three subtypes.

overall_design: AR, H3K27ac, H3K4me3 and H3K27me3 ChIP-seq data for  primary prostate carcinomas

first: Wilbert

last: Zwart

first: Yongsoo

last: Kim

first: Ekaterina

last: Nevedomskaya


title: Integrative epigenetic taxonomy of primary prostate cancer


title: The small protein MgtS and small RNA MgrR modulate the PitA phosphate symporter to boost intracellular magnesium levels

city: Bethesda

first: Gisela

last: Storz

state: MD

address: 49 Convent Drive

department: Division of Molecular and Cellular Biology

institute: NICHD

summary:
In response to low levels of magnesium (Mg2+), the PhoQP two component system induces the transcription of two convergent genes, one encoding a 31-amino acid protein denoted MgtS and the second encoding a small, regulatory RNA (sRNA) denoted MgrR. Previous studies showed that the MgtS protein interacts with and stabilizes the MgtA Mg2+ importer to increase intracellular Mg2+ levels, while the MgrR sRNA base pairs with the eptB mRNA thus affecting lipopolysaccharide modification. Surprisingly, we found overexpression of the MgtS protein also leads to induction of the PhoRB regulon. Studies to understand this activation showed that MgtS forms a complex with a second protein, PitA, a cation-phosphate symporter. Given that the additive effect of ∆mgtA and ∆mgtS mutations on intracellular Mg2+ concentrations seen previously is lost in the ∆pitA mutant, we suggest that MgtS binds to and blocks Mg2+ leakage through PitA under Mg2+-limiting conditions. Consistent with a detrimental role of PitA in low Mg2+, we also observe MgrR sRNA repression of PitA synthesis. Thus, PhoQP induces the expression of two convergent small genes in response to Mg2+ limitation whose products both repress PitA activity and levels to increase intracellular Mg2+ levels.

Funded by NIH Intramural Program ZIA HD008855-11.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Two different strains: one overexpressing mgtS (yneM) and one containing empty vector.

first: Yin

last: Xuefeng

first: Orr

last: Mona

first: Wang

last: Hanbo

first: Hobbs

last: Errett

first: Shabalina

last: Svetlana

first: Storz

last: Gisela


title: Small is big in Arabidopsis mitochondrial ribosome

city: Versailles

first: HAKIM

last: MIREAU

address: Route de Saint-Cyr

institute: INRAE

summary: Mitochondria are responsible for energy production through aerobic respiration and represent  the powerhouse of eukaryotic cells. Their metabolism and gene expression processes  combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial  gene expression processes, translation remains the most elusive. In plants, while numerous  pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their  function in translation remains unclear. Here we present the biochemical characterisation of  Arabidopsis mitochondrial ribosomes and identify their protein subunit composition.  Complementary biochemical approaches identify 19 plant specific mitoribosome proteins,  among which 10 are PPR proteins. The knock out mutations of ribosomal PPR (rPPR) genes  result in distinct macroscopic phenotypes including lethality or severe growth delays. The  molecular analysis of rppr1 mutants using ribosome profiling as well as the analysis of  mitochondrial protein levels reveal that rPPR1 is a generic translation factor, which is a novel  function for PPR proteins. Finally, single particle cryo-electron microscopy reveals the unique  structural architecture of Arabidopsis mitoribosomes, characterised by a very large small  ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted  onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially  divergent from bacterial and other eukaryote mitoribosomes, both in terms of structure and of  protein content.

overall_design: mitochondrial ribosome footprints from flowers of wildtype or PPR336 (At1g61870) mutant.

first: Tan-Trung

last: Nguyen

first: Hakim

last: Mireau


title: Integrative epigenetic taxonomy of primary prostate cancer [RNA-Seq]

overall_design: RNA-seq data for primary prostate carcinomas


title: Airway epithelial barrier, mucins and inflammasome in distinct eosinophilic, neutrophilic and mixed inflammatory phenotypes of asthma

city: Zurich

first: Ge

last: Tan

address: Winterthurerstrasse 190

department: Functional Genomics Center Zurich

institute: University of Zurich

summary:
Asthma is a complex, chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Distinct inflammatory phenotypes of eosinophilic, mixed, neutrophilic and paucigranulocytic asthma are identified in patients, but most in vivo mouse models, studying asthma mechanisms, mimic only eosinophilic phenotype in humans. The detailed unbiased in vivo studies on molecular responses among different kinds of inflammation in asthma models are lacking. Therefore, we  developed mouse models representing three different inflammatory phenotypes of airway inflammation, namely eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitisation.
We used microarrays to determine the global gene expression in the lungs of  mice with eosinophilic, mixed and neutrophilic inflammatory phenotypes to uncover underlying differences in clinical presentation and to find novel molecular targets and pathways, which might reflect different molecular mechanisms of the disease. By whole genome transcriptome profiling, we found that airway tight junction (TJ) molecules, mucins and inflammasome-related genes are differentially expressed in distinct phenotypes of allergic airway inflammation. Next, detailed analysis of several molecules from these families by quantitative RT-PCR, western blot and confocal microscopy revealed that (i) Zo-1 and Cldn18 were downregulated in all phenotypes, while Cldn4 upregulation was characteristic for neutrophilic airway inflammation; (ii) mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic asthma, and (iii) upregulation of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1 and IL-1b was characteristic for neutrophilic asthma. Finally, we showed that inflammasome/Th-17/neutrophilic axis cytokines, namely IL-1b and IL-17 impaired epithelial barrier function and increased mucins expressions in primary human bronchial epithelial cells from normal and asthmatic donors. Our findings suggest that differential expression of TJs, mucins and inflammasome-related molecules in distinct asthma phenotypes could be mechanistically linked and might further reflect the differences observed in the clinic.

overall_design:
Different asthma phenotypes - eosinophilic, mixed, and neutrophilic, were induced in Balb/c mice via different ways of house dust mite (HDM) sensitisation and compared to the non-sensitized control group. For eosinophilic asthma mice were sensitized by three intraperitoneal (i.p.) injections of HDM extract containing 0.75 DU Der f and 0.75 DU Der p (ALK-Abelló A/S) adsorbed to 1.5 mg Al(OH)3 (Pierce, Rockford, IL, USA)  in 200 µl PBS on days 0, 7 and 21. On days 26 to 28, mice were challenged intranasally (i.n.) with the similar dose of HDM extract dissolved in 50 µl PBS. For induction of the mixed or neutrophilic endotype mice were i.n. sensitized/challenged with the HDM extract (0.75 DU Der f /0.75 DU Der p or 7.5 DU Der f/7.5 DU Der p, respectively) in 50 µl PBS once a week a total of four times  Intranasal application of only PBS served as a control. Whole lungs total RNA was used for transcriptomics from 3 mice in each phenotype group and control group. Lung samples were also snap frozen in liquid nitrogen for further analyses by immunofluorescence staining, Western blotting and mRNA expression. All animal procedures were performed with approval from the University of Marburg local authorities (Regierungspräsidium Gießen).

>>>CEL files are not available for this series<<<

first: Holger

last: Garn

first: Milena

last: Sokolowska


title: Heterogeneity of Ly6G+Ly6C+ myeloid-derived suppressor cell (MDSC) infiltrates during S. aureus biofilm infection

city: Omaha

first: Sean

last: West

state: NE

institute: University of Nebraska

summary: The two immune cell populations Myeloid-derived suppressor cells (MDSCs), monocytes (MONO) and neutrophils (PMNs) are difficult to differentiate because of shared surface marker expression. Here we utilize the integrin receptor CD11b combined with conventional Ly6G and Ly6C expression to more accurately separate cellular populations via FACS. Then we apply high-throughput RNA Sequencing to Ly6G+Ly6C+CD11bhigh MDSC, Ly6G+Ly6C+CD11blow PMN and Ly6G-Ly6C+ monocyte populations. A total of 6,466 genes were significantly differentially expressed in MDSCs vs. monocytes, whereas only 297 genes were significantly different between MDSCs and PMNs. A number of genes implicated in cell cycle regulation were identified, and in vivo EdU labeling revealed that over 75% of MDSCs proliferated locally at the site of S. aureus biofilm infection.

overall_design: RNA-Seq of myeloid-derived suppressor cells (MDSCs), neutrophils (PMNs), and monocytes during S. aureus biofilm infection in mice

first: Heim

last: Cortney

first: West

last: Sean

first: Ali

last: Hesham

first: Kielian

last: Tammy


title: Kinetics of LPS induction in wt and Cop1-ko BMDMs

city: South San Francisco

first: Sarah

last: Kummerfeld

address: 1 DNA Way

institute: Genentech

summary:
Bone marrow was extracted from mice that are COP1-wt Rosa26-CreERT2 or COP1-floxed Rosa26-CreERT2 BMDMs were obtained by culturing bone marrow precursors in media containing 20% of supernatant from L929 cells. At day 4 of differentiation 4-OHT was added at 1uM to induce deletion of COP1 in BMDMs derived from COP1-floxed mice. At day 7 of differentiation, BMDMs were treated with 100 ng/ml of LPS or not. BMDMs were directly harvested in lysis buffer (from Qiagen RNeasy mini kit) at different time points (0h, 2.5h, 2.5h, 4h, 6h, 9h and 13h) following LPS stimulation.
Three BMDMs preparations per group:
G1: BMDMs from COP1-wt mice (expressing the wt allele of COP1) CRE positive.
G2: BMDMs from COP1-floxed mice (expressing the floxed allele of COP1) CRE positive

overall_design: RNA from BMDMs following LPS treatment at different time points from mice that are COP1-wt Rosa26-CreERT2 or COP1-floxed Rosa26-CreERT2.

first: Ada

last: Ndoja


title: Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer

city: Indianapolis

first: Xiongbin

last: Lu

state: Indiana

address: 980 W. Walnut Street, R3-C218D	MMGE

institute: Indiana University School of Medicine

summary: Amplification of chromosome 17q23 is a frequent genomic event that occurs in ~ 11% of human breast cancers. The 17q23 amplification is enriched in HER2+ breast cancers, which is significantly correlated with poor clinical outcomes. Previous studies have identified the oncogenic phosphatase WIP1 gene in the amplicon, which functions as a master inhibitor in DNA damage response. While the possibility of any other protein-coding oncogenes in the WIP1-containing 17q23 amplicon was ruled out, our analysis of human breast cancer genomics uncovered an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing amplicons. Interestingly, DEAD-box helicase 5 (DDX5), co-amplified with WIP1 and MIR21 in the 17q23 amplicon, facilitates the essential processing of primary miR-21 transcripts. Accordingly, the 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 using small molecular inhibitor against WIP1 (GSK2830371) and anti-miR-21 oligonucleotides selectively inhibits the proliferation, survival and tumorigenic potential of HER2+ breast cancer cells harboring 17q23 amplification. However, the in vivo bioavailability of the two agents in their free form is poor. To overcome the resistance of trastuzumab-based therapies in vivo, we developed pH-sensitive nanoparticles for specific co-delivery of the two agents into breast tumors. The nanoparticles consist of four materials approved by the Food and Drug Administration (FDA) for medical use: Poly (d,l-lactide-co-glycolide) (PLGA), Pluronic F127 (PF127), chitosan, and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC). Moreover, chitosan was modified with guanidine to form chitosan-guanidine (CG), which not only can improve the encapsulation efficiency of anti-miR-21 oligonucleotides but also effectively capture carbon dioxide (CO2) into the nanoparticle to achieve the ‘nano-bomb’ effect for triggered drug release under the reduced pH in tumors. The two agents (inhibitors of miR-21 and WIP1)-laden nanoparticles can be used to efficiently kill trastuzumab-resistant HER2+ breast cancer cells, leading to a profound reduction of the tumor growth in vivo. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the HER2+ breast cancers resistant to anti-HER2-based therapies.

overall_design: Primary MMTV-ErbB2 mouse mammary epithelial cells (8-wk old) were isolated as previously reported and then total mRNA was extracted using Direct-zol RNA extraction kit (duplicates, Zymo Research) and then submitted for deep sequencing. Deep sequence data were mapped, normalized and the differentially expressed genes were collected for pathway analysis.

first: Yunhua

last: Liu

first: Jiangsheng

last: Xu

first: Hyun

last: Choi

first: Cecil

last: Han

first: Yuanzhang

last: Fang

first: Yujing

last: Li

first: Kevin

last: Van der Jeught

first: Hanchen

first: Lu

last: Zhang

first: Michael

last: Frieden

first: Lifei

last: Wang

first: Haniyeh

last: Eyvani

first: Yifan

last: Sun

first: Gang

last: Zhao

first: Yutian

first: Sheng

first: Jun

last: Wan

first: Cheng

last: Huang

first: Guang

last: Ji

first: Xiaoming

last: He

first: Xinna


title: Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics - mouse

first: Darya

last: Vanichkina

summary: MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3cyanovinylcarbazole ( CNV K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNV K-containing miR-29b ( CNV K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, arehighly sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNV K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and findrobust conservation of the focal adhesion and apoptotic target pathways in mouse and mouse. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNV K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNV K-containing small RNA mimics may find applicability in other experimental models

overall_design: pulldown assay with 3x3x3x3 design: miRNA mimic and scrambled miRNA mimic x input and pulldown conditions. Nuclear/cytoplasmic fractionation (1 replicate x nuc/cyto x 4 conditions)

first: Anupma

last: Choudhary


title: Mettl3-mediated m6A RNA methylation  regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

city: Chengdu

first: Quan

last: Yuan

state: Sichuan

address: 14 Third section, Renmin Nan Road

institute: West China Hospital of Stomatology, Sichuan University

summary: The aim is to analyze the transcriptome profiling (RNA-seq) of Mettl3 knockout mesenchymal stem cells (MSCs) and identify m6A modified mRNAs (m6A-seq). For RNA-seq, total RNAs from MSCs of Prx1-Cre;Mettl3fl/fl mice and their control littermates were extracted and purified using poly-T oligo-attached magnetic beads. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations, and were then subjected to Illumina HiSeq 2500. We used FastQC (v0.11.5) and FASTX toolkit (0.0.13) to control the quality of RNA-seq data and mapped them to Mus musculus reference genomes using HISAT2 (v.2.0.4). Ballgown software (v.3.4.0) was performed to identify differentially expressed genes and transcripts. Genes were considered significantly differentially expressed if showing ≥ 2.0 fold change and P value < 0.05. For m6A-seq, total RNAs were isolated from MSCs and then mRNAs were purified using poly-T oligo-attached magnetic beads. The purified mRNAs were fragmentated and used for immunoprecipitation with anti-m6A antibody, then the pulled down fragments were purified and used for libraries construction with the Illumina TruSeq Stranded mRNA Sample Prep Kit following the manufacturer's instructions. m6A libraries were sequenced with Illumina NextSeq500. Sequencing reads were aligned to mm9 using tophat and peak calling was performed with MACS2.

overall_design: The mRNA profiles of WT and Prx1-Cre;Mettl3fl/fl  MSCs were generated by deep sequencing, in duplicate, using Illumina HiSeq 2500.  m6A profiles of WT MSCs were sequenced with Illumina NextSeq500.


title: PyMT-1099, a murine derived cell line is a breast cancer specific model for EMT

city: Basel

first: Ravi Kiran Reddy

last: KALATHUR

state: Basel

address: Mattenstrasse 28

department: Departement Biomedizin

institute: University of Basel

summary: Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. However, several of the studies have been carried out using model systems that don’t appropriately recapitulate all stages of the dynamic process of EMT. Hence, there is a need to overcome this limitation by development of a model system that allows us to mimic each stage of EMT and accurately assess the plastic changes associated with it.   Methods: We have derived a cancer cell line from the PyMT-MMTV model of breast cancer, named PyMT-1099 cells, and undertaken a detailed characterization of the morpho-genetic changes it undergoes during a TGF-induced EMT. Further, we have also performed high throughput transcriptomics on PyMT-1099 cells undergoing EMT in a high resolution kinetic of TGF treatment.  Results: We show that PyMT-1099 cells undergo an EMT comparable to the classically used immortalized NMuMG cells as assessed by morphological, and marker expression changes on TGFtreatment. Further, PyMT-1099 cells can also migrate in vitro in response to TGF treatment. These cells are also tumorigenic and lead to metastasis formation when transplanted into immunocompromised mice.  Conclusion: In this study we report the development of PyMT-1099 cells as an excellent tool to model and study breast cancer-associated EMT both in vitro and in vivo  and show that these cells overcome the limitations posed by other cellular systems currently being used to study EMT

overall_design: RNA-Seq of TGFb-induced EMT and MET over a timecourse of 10 days in PyMT-1099 cells performed in biological duplicates

first: Meera

last: Saxena

first: Ravi

last: Kalathur

first: Gerhard

last: Christofori


title: TRPS1 is a lineage-specific transcriptional dependency in breast cancer [methylation array]

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

type: Methylation profiling by genome tiling array

summary: We performed an unbiased cell viability-based pooled shRNA screen on 59 cell lines to identify novel epigenetic and transcriptional dependencies of multiple cancer types, including leukemia, neuroblastoma, breast, colorectal, prostate, and rhabdoid tumors. Here, we identified Tricho-Rhino-Phalangeal Syndrome Type I protein (TRPS1) as one of the most significant hits specific for breast cancer cell lines. Downregulation of TRPS1 resulted in cell cycle arrest and apoptosis increase in vitro and impaired tumorigenic capacity in vivo. We characterized TRPS1 genomic targets and protein interactome. We identified GATAD2B as an important partner of TRPS1, uncovering novel epigenetic network crucial for breast cancer cell survival.

overall_design: DNA methylation: Array based methylation analysis of 59 cell lines.

first: Robert

last: Witwicki

first: Muhammad

last: Ekram

first: Xintao

last: Qiu

first: Michalina

last: Janiszewska

first: Shaokun

last: Shu

first: Mijung

last: Kwon

first: Anne

last: Trinh

first: Elizabeth

last: Frias

first: Nadire

last: Cochran

first: Greg

last: Hoffman

first: Yingtian

last: Xie

first: Gregory

last: McAllister

first: Rob

last: McDonald

first: Javad

last: Golji

first: Michael

last: Schlabach

first: Kavitha

last: Venkatesan

first: Antoine

last: deWeck

first: Nicholas

last: Keen

first: Ho

last: Chan

first: David

last: Ruddy

first: Tomas

last: Rejtar

first: Sosathya

last: Sovath

first: Serena

last: Silver

first: William

last: Sellers

first: Zainab

last: Jagani

middle: D

last: Hogarty

first: Ramesh

middle: A

last: Shivdasani

first: Charles

last: Roberts

first: Myles

last: Brown

first: Kimberly

last: Stegmaier

first: Henry

last: Long

last: Pellman


title: Mesenchymal Epithelial Transition (MET) Timecourse of NMuMG-E9 cells treated with TGFb

first: Ravi Kiran Reddy

last: KALATHUR

summary:
Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. However, several of the studies have been carried out using model systems that don’t appropriately recapitulate all stages of the dynamic process of EMT. Hence, there is a need to overcome this limitation by development of a model system that allows us to mimic each stage of EMT and accurately assess the plastic changes associated with it.

Methods: We have derived a cancer cell line from the PyMT-MMTV model of breast cancer, named PyMT-1099 cells, and undertaken a detailed characterization of the morpho-genetic changes it undergoes during a TGFbeta-induced EMT. Further, we have also performed high throughput transcriptomics on PyMT-1099 cells undergoing EMT in a high resolution kinetic of TGFbeta treatment.

Results: We show that PyMT-1099 cells undergo an EMT comparable to the classically used immortalized NMuMG cells as assessed by morphological, and marker expression changes on TGFbeta treatment. Further, PyMT-1099 cells can also migrate in vitro in response to TGFbeta treatment. These cells are also tumorigenic and lead to metastasis formation when transplanted into immunocompromised mice.

Conclusion: In this study we report the development of PyMT-1099 cells as an excellent tool to model and study breast cancer-associated EMT both in vitro and in vivo  and show that these cells overcome the limitations posed by other cellular systems currently being used to study EMT

overall_design: RNA-Seq of MET over a timecourse of 10 days on withdrawl of TGFbeta in NMuMG (E9) cells performed in biological duplicates

first: Meera

last: Saxena

first: Gerhard

last: Christofori

first: Ravi

last: Kalathur


title: mRNA sequencing analysis of transcripts from hippocampus of Wt1∆ mice

status: Public on Oct 02 2018

city: NEW YORK

first: Chiara

last: Mariottini

address: 1425 Madison Ave, Room 12-52

department: Pharmacological Sciences

institute: Icahn School of Medicine at Mount Sinai

summary:
Purpose: Study the differentially expressed genes in the hippocampus of mice, comparing Wt1∆ mice with their control wild type littermates in basal conditions (naïve untrained animals).
Methods: Forebrain-specific deletion of Wt1 was achieved by crossing animals homozygous for the conditional Wt1 knockout allele (Wt1fl/fl) with a transgenic line, Camk2a-Cre, (B6.Cg-Tg(Camk2a-cre)T29-1Stl/J; Jackson Lab: http://jaxmice.jax.org/strain/005359.html). Expression of Cre recombinase resulted in the in-frame deletion of exons 8 and 9 and generated a truncated allele encoding a shortened non functional WT1 protein lacking zinc fingers 2 and 3 in their forebrain (starting at P21). Brains of Wt1∆ mice (n=2) and their control littermates (wild type; n=2) were removed, dorsal hippocampi were dissected and used for library preparation. The seq library was prepared using TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA).
For the RNA-Seq data analysis Tophat 2.0.13, bowtie 2.1.0 , samtool 0.1.7 and cufflinks 1.3.0  were used. The rn5-bowtie2 index was generated with the command 'bowtie2-build rn5.fa rn5'. The 'rn5.fa'-file was downloaded from the UCSC genome browser. The mm10-bowtie2 index was downloaded from http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml.  RefSeq geneTracks and GTF-files for the rn5 and mm10 genome assembly were downloaded from UCSC genome browser. Common gene ids in the GTF-files were matched to individual transcript_ids using the corresponding official symbols obtained from the geneTracks files.
Conclusions: Our study illustrates the nature of the different transcripts in Wt1∆ mice compared to their control wild type littermates at basal state (untrained). This experiment allowed us to identify wich genes are modulated in the hippocampus by the transcription fator WT1.

overall_design: mRNA profiles  from dorsal hippocami Wt1∆ mice and control wild type littermates were generated by deep sequencing using TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA).

first: Jens

last: Hansen

first: Leonardo

last: Munari


title: The Scalloped and Nerfin-1 transcription factors cooperate to maintain neuronal cell fate

city: Clayton

first: Jan

last: Schroeder

state: VIC

address: Building 75 Innovation Walk

department: Anatomy & Developmental Biology

institute: Monash University

summary: The ability of cells to stably maintain their fate is governed by specific transcription regulators. Here, we show that the Scalloped (Sd) and Nervous fingers-1 (Nerfin-1) transcription factors physically and functionally interact to maintain medulla neuron fate in the Drosophila melanogaster CNS. Using Targeted DamID we find that Sd and Nerfin-1 occupy a highly overlapping set of target genes, including regulators of neural stem cell and neuron fate, and signalling pathways that regulate CNS development such as Notch and Hippo. Modulation of either Sd or Nerfin-1 activity causes medulla neurons to dedifferentiate to a stem cell-like state and this is mediated at least in part by Notch pathway deregulation. Intriguingly, orthologues of Sd and Nerfin-1 have also been implicated in control of neuronal cell fate decisions in both worms and mammals. Our data indicate that this transcription factor pair exhibits remarkable biochemical and functional conservation across metazoans.

overall_design: DamID-seq on CNSs from wandering third instar Drosophila larvae expressing Dam control, Dam-Sd or Dam-Nerfin-1 transgenes driven by GMR31H08-Gal4 (two replicates each). ATACseq and RNAseq on FACS sorted wildtype Nerfin-GFP expressing neurons (3 replicates each).

first: Joseph

last: Vissers

first: Francesca

last: Froldi

first: Anthony

last: Papenfuss

first: Louise

last: Cheng

first: Kieran

middle: F

last: Harvey


title: mRNA-seq of immortailized bone marrow derived macrophages (iBMDM) after vesicular stomatitis virus (VSV) infection

city: BEIJING

first: Peiyan

last: Wang

state: 北京

address: NO.38 ROAD XUEYUAN, DISTRICT HAIDIAN, BEIJING, CHINA, 北京市海淀区学院路38号北京大学医学部

institute: Peking University

summary: Analysis of mRNA-seq reveals how wild type immortalized bone marrow derived macrophages response to VSV infection

overall_design: Immortalized bone marrow derived macrophages were infected by VSV for 0 or 6 hours


title: A multiplexed assay for exon recognition reveals that an unappreciated fraction of rare genetic variants cause large-effect disruptions to splicing

city: Los Angeles

last: Insigne

state: CA

department: Biochemistry

institute: UCLA

summary: Mutations that cause exon skipping can have severe consequences on gene function and cause disease. Here we explore how human genetic variation affects exon recognition by developing a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter, and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants, even outside the context of disease, can have large functional effects at appreciable rates, and that MFASS enables their empirical assessment for large-effect splicing defects at scale.

overall_design: MFASS is a splicing minigene reporter. There are three synthetic oligo libraries, SRE library in two backbones and the SNV library. The oligo library is cloned in between a split GFP reporter with a downstream mCherry reporter. Exon inclusion leads to both GFP and mCherry fluorescence, exon skipping leads to only mCherry fluorescence. We sort the libray of cells based on these two fluorescent reporters using fluorescence activated cell sorting (FACS). We then sequence each individual bin to get a quantitiative measure of splicing.  For the SRE library, we sort into three bins, with two replicates. Double positive (DP) indicates both GFP and mCherry are present and correspond to exon skipping. Intermediate (INT) indicates intermediate levels of GFP and mCherry. Single positive (SP) indicates no GFP present but mCherry present, corresponds to exon inclusion. For the SNV library, the sorting is similar except there are four bins, also done in replicate. Hi indicates both GFP and mCherry, Int-Hi and Int-Lo correspond to two intermediate bins, and Lo corresponds to no GFP present but mCherry present. We use the read counts across all bins, present in processed data files, to calculate an inclusion index for an individual sequence.

first: Rocky

last: Cheung

first: David

last: Yao

first: Christina

middle: P

last: Burghard

first: Jeffrey

first: Yun-Hua

last: Hsiao

first: Eric

middle: M

last: Jones

first: Daniel

last: Goodman

first: Xinshu

last: Xiao

first: Sriram

last: Kosuri


title: Next Generation Sequencing of Wild Type and PTPRO-/- ESCC tumoral Transcriptomes

city: Guangzhou

first: Hong

middle: mei

last: Dong

address: 601 Huangpu Avenue West

department: Institute of Precision Cancer and Pathology

institute: Jinan University Medical College

summary: To induce tumorigenesis in the esophagus, 100 mg/mL of the water-soluble carcinogen 4NQO (Sigma-Aldrich) was added to the drinking water of 6-week-old mice for 16 weeks. The mice were sacrificed when body weight loss >1/3; otherwise, they were sacrificed 12 weeks after completion of 4NQO treatment.Total RNA from murine esophageal squamous cell carcinoma (Ptpro-/- vs. Ptpro+/+, N=3/group) was extracted and purified, transcriptome profiles were generated by deep sequencing, using Illumina HiSeq 2500 sequencer.

overall_design: Transcriptome profiles of esophageal squamous cell carcinoma of 34-week old mice (Ptpro-/- vs. Ptpro+/+, N=3/group) were generated by deep sequencing, using Illumina HiSeq 2500 sequencer.

first: Hao

last: Zhang

first: Hongmen


title: Gene expression analysis of Trem1-deficient Kupffer cells subjected to carbon tetracloride (CCl4) in liver fibrogenesis.

city: augusta

first: Anatolij

last: Horuzsko

state: Georgia

address: 1410 Laney Walker Boulevard

institute: georgia cancer center

summary: Analysis of Kupffer cells at gene expression level. The hypothesis tested in the present study was that the expression of TREM-1 on Kupffer cells isolated from wild type mice subjected to single dose of carbon tetracloride and sacrificed 12 hours after injection influences the expression of multiple genes implicating in several signaling pathways. Results provide important information of the response of Trem1-sufficient and Trem1-deficient Kupffer cells to carbon tetracloride, such as inflammatory responsive genes, up-or-down regulated inflammation, acute liver injury and liver fibrogenesis.

overall_design: Total RNA isolated from purified Kupffer cells of Trem1-deficient mice injected with single dose of carbon tetracloride and sacrifced 12 hours after and compared to purified Kupffer cells of wild type mice treated with single dose of oil (control) or carbon tetracloride and sacrificed 12 hours after.

first: Amiran

last: Dzutsev

first: Anh

last: Nguyen-Lefebvre


title: The transcription factor Foxp1 preserves integrity of an active Foxp3 locus in extrathymic Treg cells

city: Cheonan

first: Keunsoo

last: Kang

address: 119 Dandae-ro

country: South Korea

institute: Dankook University

summary: In order to identify if there is any role of Foxp1 in the DNA demethylation of Foxp3 enhancer regions of induced Treg (iTreg) cells we performed bisulfite sequence analysis of Foxp1 deficient in vitro generated iTreg cells and compared their methylation status after 7 days of iTreg induction, at which point Foxp1 is deleted in the iTreg cells derived from Foxp3YFPCreFoxp1f/f  (KO) mice.

overall_design: Examination of methylation in 3 loci on the X chromosome by bisulfite sequencing protocol using in vitro generated itreg cells.

first: Sayantani

last: Ghosh

first: Sinchita

last: Roy-Chowdhuri

first: Sin-Hyeog

last: Im

first: Dipayan

last: Rudra


title: Aberrant splicing in B-cell acute lymphoblastic leukemia [B-ALL]

city: Philadelphia

first: Andrei

last: Thomas-Tikhonenko

state: PA

address: 3501 Civic Center Blvd, CTRB 4056

department: Children's Hospital of Philadelphia

institute: Perelman School of Medicine at the University of Pennsylvania

summary: Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing them to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ~100 SFs, e.g. hnRNPA1. hnRNPA1 3’UTR was most pervasively misspliced, yielding the transcript  subject to nonsense-mediated decay. Thus, we knocked it down in B-lymphoblastoid cells, identified 213 hnRNPA1-dependent splicing events, and defined the hnRNPA1 splicing signature in pediatric leukemias. One of its elements was DICER1, a known tumor suppressor gene; its LSVs involved the 5’ UTR, suggestive of splicing as a mechanism of translational deregulation. Additionally, we searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 genes. 77 LSVs were confirmed using two large independent B-ALL RNA-seq datasets. In fact, the twenty most common B-ALL drivers showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contribute to disease pathogenesis.

overall_design: We profiled 18 pediatric B-cell acute lymphoblastic leukemias

first: Kathriyn

middle: L

last: Black

first: Ammar

middle: S

last: Naqvi


title: Aberrant splicing in B-cell acute lymphoblastic leukemia [cell line]

overall_design: We profiled hnRNPA1 Ctrl and hnRNPA1 knockdown with 2 replicates each.

first: Kathryn


title: RNA-Seq analysis of skin from bleomycin induced scleroderma murine models treated with EHP-101

city: Córdoba

first: Eduardo

last: Muñoz

address: Avda Menéndez Pidal s/n

country: Spain

institute: Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), University of Córdoba

summary: Systemic sclerosis (SSc) or scleroderma is a chronic multiorgan autoimmune disease of unknown etiology characterized by vascular, immunological and fibrotic abnormalities. Several lines of evidence have shown that the endocannabinoid system (ECS) may play a role in the pathophysiology of SSc. VCE-004.8, a CBD aminoquinone derivative, is a dual PPARγ/CB2 that alleviates bleomycin (BLM)-induced skin fibrosis. Herein we report that EHP-101, an oral lipidic formulation of VCE-004.8, prevents skin and lung fibrosis and collagen accumulation in BLM challenged mice. Immunohistochemistry analysis of the skin demonstrate that EHP-101 prevents macrophage infiltration, and the expression of Tenascin C (TNC), VCAM, and the α-smooth muscle actin (SMA). In addition, a reduced expression of vascular CD31, paralleling skin fibrosis, was also prevented by EHP-101. RNAseq analysis in skin biopsies showed a clear effect of EHP-101 in the inflammatory and epithelial-mesenchymal transition transcriptomic signatures. TGF-beta regulated genes such as matrix metalloproteinase-3 (Mmp3), cytochrome b-245 heavy chain (Cybb), lymphocyte antigen 6E (Ly6e), vascular cell adhesion molecule-1 (Vcam1) and the Integrin alpha-5 (Itga5) were induced in BLM mice and repressed by EHP-101 treatment. We also intersected differentially expressed genes in EHP-101-treated mice with dataset of human scleroderma intrinsic genes and found 53 overlapped genes, including the C-C motif chemokine 2 (Ccl2) and the interleukin 13 receptor subunit alpha 1 (IL-13Ra1) genes, which have been studied as biomarkers of SSc. Altogether the results indicate that this synthetic cannabinoid qualifies as a novel compound for the management and possible treatment of scleroderma and, potentially, other fibrotic diseases.

overall_design: RNA-Seq profiles were generated for six- to eight-week-old female BALB/c mice in three conditions: Control, Bleomycin and Bleomycin + EHP-101 treatment (N=2).

first: Adela

last: García-Martín

first: Martín

last: Garrido-Rodríguez

first: Carmen

last: Navarrete

last: del Río

first: María

last: Bellido

first: Giovanni

last: Appendino

first: Marco

last: Calzado


title: XBP1s Activation Globally Remodels N-Glycan Structure Distribution Patterns

city: Cambridge

first: Charles

middle: Arthur

last: Whittaker

institute: Koch Institute

summary: The unfolded protein response (UPR), as its name implies, safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated, XBP1s-mediated transcriptional response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s’ transcriptional output for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional consequence of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. XBP1s activity decreases sialylation of tri- and tetra-antennary N-glycans in the HEK293 membrane proteome and secretome, while substantially increasing the population of high mannose N-glycans only in the secretome. Related, but distinctive, signatures in the HEK293 N-glycome are observed when the entire UPR is activated in a stress-dependent manner using thapsigargin. In HeLa cells, stress-independent XBP1s activation increases the population of cell surface high mannose N-glycans and tetra-antennary N-glycans. mRNA profiling experiments suggest that the XBP1s-mediated remodeling of the N-glycome may re-flect a coordinated consequence of transcriptional resculpting of the N-glycan maturation pathway by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s is a master regulator of N-glycan maturation. Moreover, because the sugars on cell surface proteins or on those proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling pathways into al-tered interactions with the extracellular environment.

overall_design: Three biological replicates of HeLaXBP1s cells treated with DMSO vehicle, 1 ug/ml dox or 750 nM Thapsigargin.

first: Madeline

middle: Y

last: Wong

first: Kenny

last: Chen

first: Aristotelis

last: Antonopoulos

first: Brian

middle: T

last: Kasper

first: Mahender

middle: B

last: Dewal

first: Rebecca

last: Taylor

first: Pyae

last: Hein

first: Anne

last: Dell

first: Stuart

last: Haslam

first: Lara

middle: K

last: Mahal

first: Matthew

last: Shoulders


title: Transcriptome analysis of the HepG2 cells expressing hepatic transcription factors

city: Fukuoka

first: Atsushi

last: Suzuki

state: Fukuoka

address: 3-1-1 Maidashi, Higashi-ku

department: Medical Institute of Bioregulation

institute: Kyushu University

summary: In order to elucidate an anticancer function of some hepatic transcription factors (TFs), we induced the three hepatic TFs: HNF1A, HNF4A, and FOXA3, into a hepatocellular carcinoma cell line, HepG2, with some combination.

overall_design: RNA-Seq for HepG2 cell with control EGFP, HNF1A, HNF4A, FOXA3, and a combination of the three TFs at day 7 and 14 after transduction.

first: Yasuyuki

last: Ohkawa

first: Miyako

last: Udono

first: Kenichi

last: Horisawa

first: Yasuo

last: Takashima


title: The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression

city: Milan

first: Jose

middle: Manuel

last: Garcia-Manteiga

address: Via Olgettina 58

department: Center for Translational Genomics and Bioinformatics

country: Italy

institute: OSR - San Raffaele Scientific Institute

summary: Myoblast fusion (MF) is a complex process required for muscle formation. During development, post-natal muscle growth and adult muscle regeneration, myoblasts proliferate and fuse to generate multinucleated fibers. MF defects have been described in an increasing number of muscle diseases. Moreover, MF plays also a relevant role in the delivery of therapeutics to muscle fibers. Hence, the identification of novel MF activators bears strong therapeutic relevance. Despite the central role of MF, the mechanisms governing this process are incompletely understood, and no epigenetic regulator has ever been described.  Ash1L is a histone methyltransferase responsible for H3K36me2 and a member of the Trithorax group of epigenetic activators. It is involved in several diseases, including FSHD muscular dystrophy, autism and cancer. Nevertheless, its physiological role in skeletal muscle is unknown.  For the first time, we found that Ash1L expression is regulated during skeletal muscle development, positively correlated with that of key MF genes and reduced in Duchenne muscular dystrophy. During physiological muscle differentiation or muscle regeneration, Ash1L shows a peak of expression coincident with the activation of MF. Loss-of-function experiments support a selective and evolutionary conserved requirement for Ash1L in MF. Accordingly, Ash1L KO mice display small and underdeveloped skeletal muscles due to a MF defect. By combining RNA- and ChIP-sequencing, we identified direct Ash1L targets in muscle. Intriguingly, nearly all of them are known Polycomb target genes suggesting that Ash1L is required to counteract Polycomb repressive activity to allow activation of key myogenesis genes. In particular, we found that Ash1L drives MF by directly activating the expression of the key MF gene Cdon. Our results promote Ash1L as the first epigenetic regulator of MF and suggest that its activity could be targeted to improve cell therapy for muscle diseases.

overall_design: Proliferating C2C12 cells were transfected with non targeting (NS) or Ash1L siRNAs (Ash1L KD) and collected after 24hours (myoblasts) or after three days of differentiation in mitogen-poor medium (myotubes). For RNA-seq assay, we collected both myoblasts and myotubes from three indepentent experiments. For ChIP-seq analysis, we collected C2C12 myoblasts at day 1 of differentiation from three indepentent experiments.

first: Ilaria

last: Castiglioni

first: Davide

last: Gabellini