title: Patient- and Cell Type-Specific Heterogeneity of Metformin Response.

city: Rochester

first: Michael

last: Asiedu

address: 200 Fisrt ST SW

department: Surgery

institute: Mayo Foundation for Medical Education and Research

summary:
Most FDA approved drugs are not equally effective in all patients, suggesting that identification of biomarkers to predict responders to a chemoprevention agent will be needed to stratify patients and achieve maximum benefit. The goal of this study was to investigate both patient specific and cell-context specific heterogeneity of metformin response, using cancer cell lines fibroblast cell lines and induced pluripotent stem cells differentiated into lung epithelial lineages.
We performed  transcriptome analysis on both patient-derived fibroblast cell lines and cancer cell lines to assess differential metformin response and identify response genes. We found differences in response to metformin treatment across a variety of cell lines and cellular contexts, suggesting heterogeneity that may be patient and cell-type specific. Gene expression profiling and analysis of metformin sensitive and resistant cells identified differentially expressed genes that may be able to stratify patients into metformin responders and non-responders.

overall_design: Patient derived fibroblast cells, differentiated IPS cells and cancer cells were each treated with their respective IC50 concentrations of metformin and DMSO for 72 hours. Total RNA was extracted,  hybridized to the Affymetrix Gene Chip Human Genome U133 Plus 2.0 single arrays and using GeneChip Scanner 3000. Principle Component Analysis (PCA) and unsupervised hierarchical clustering were used to perform quality checks on the samples. Samples from control and treated, and from resistant and sensitive groups were compared using 1-way ANOVA (analysis of variance). Significantly changed genes obtained from ANOVA were prioritized by a combination of P value and fold change.

first: Dennis

last: Wigle


title: Dynamics of Telomere Rejuvenation During Chemical Induction to Pluripotent Stem Cells

status: Public on May 01 2018

city: Tianjin

first: Haifeng

last: Fu

address: Weijin Road Num. 74

country: China

institute: Nankai University

type: Expression profiling by high throughput sequencing

summary: Chemical induced pluripotent stem cells (CiPSCs) have been successfully achieved and may provide an alternative and attractive source for stem cell-based therapy. Sufficient telomere lengths are critical for unlimited self-renewal and genomic stability of pluripotent stem cells. Dynamics of telomere reprogramming and whether and how telomeres are sufficiently elongated in the CiPSCs have remained to be understood. We show that CiPSCs acquire telomere lengthening with increasing passages after clonal formation. Both telomerase activity and recombination-based mechanisms are involved in the telomere elongation. Telomere lengths strongly indicate the degree of reprogramming, pluripotency and differentiation capacity of CiPSCs. Nevertheless, telomere damage and shortening occur at late stage of lengthy induction, limiting CiPSC formation. Recombination mechanism is not activated during induction until CiPSC clonal formation and passages. We find that histone crotonylation induced by crotonic acid can activate two-cell genes including Zscan4, alleviate telomere damage and shortening during induction and promote CiPSC generation. Moreover, crotonylation decreases abundance of heterochromatic H3K9me3 and HP1a at subtelomeres and Zscan4 loci. Taken together, telomere rejuvenation links to reprogramming and pluripotency of CiPSCs. Crotonylation facilitates telomere maintenance and enhances chemical induced reprogramming to pluripotency.

overall_design: We collected day 0, day 15, day 30, day 40 reprogramming cells and CiPSCs to detect telomeres dynamic. We collected day 20, day 24 and day 28 reprogramming cells with or without crotonic acid to do RNA-sequence experiment to analyze how CA can promote CiPSCs generation. We collected CiPS cell lines by three induction methods to analyze these cell lines transcriptome.

first: Cheng-lei

last: Tian

first: Xiaoying

last: Ye


title: Uhrf1 regulates active transcriptional marks at bivalent domains in pluripotent stem cells through Setd1a

status: Public on May 02 2018

city: New Haven

first: Kun-Yong

last: Kim

state: CT

address: 10 Amistad Street

department: Genetics

country: USA

institute: Yale University

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Identification of Nrf2 regulated genes by RNA sequencing

city: aarhus

first: Yonglun

last: Luo

address: Wilhelm Meyers Allé 4

country: Denmark

institute: Aarhus University

summary: The transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is activated by the metabolite  itaconate during metabolic reprogramming. Activated Nrf2 then dampens the release of pro-inflammatory  cytokines and type I IFNs in response to toll-like receptor stimulation. If and how Nrf2 affects cytosolic  antiviral sensing and whether this occurs during metabolic reprogramming is currently not known. Here, we  show that Nrf2 is a negative regulator of the adaptor molecule STimulator of INterferon Genes (STING),  which signals downstream of the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS). The regulation  of STING by Nrf2 was inducible by the metabolite itaconate, specific to human cells, and sufficient to  decrease the responsiveness to STING agonists and to increase the susceptibility to infection with DNA  viruses. Mechanistically, Nrf2 regulated STING expression post-transcriptionally by increasing STING  mRNA stability. Lastly, treatment with itaconate or with the chemical Nrf2 inducer sulforaphane repressed  STING expression and the release of type I IFNs in cells from patients with the STING dependent  interferonopathy SAVI. With this report we identify Nrf2 as an important regulator of cGAS-STING  signaling pathway and link metabolic reprogramming to control of cytosolic DNA sensing.

overall_design: RNA sequencing of control and Nrf2 siRNA treated A549 cells was generated by Active Motif (Carlsbad, California, USA), in triplicate, Illumina HiSeq 4000.

first: David

last: Olagnier

first: Aske

middle: M

last: Brandtoft

first: Camilla

last: Gunderstofte

first: Nikolaj

middle: L

last: Villadsen

first: Christian

last: Krapp

first: Anne

last: Thielke

first: Anders

last: Laustsen

first: Suraj

last: Peri

first: Anne-Louise

last: Hansen

first: Lene

last: Bonefeld

first: Jacob

last: Thyrsted

first: Lin

last: Lin

first: Victor

last: Bruun

first: Marie

middle: B

last: Iversen

first: Virginia

last: Artegoitia

first: Rongtuan

first: Siddharth

last: Balachandran

first: Mette

last: Nyegaard

first: Bernadette

last: Marrero

first: Raphaela

last: Goldbach-Mansky

first: Luke

last: O’Neill

first: Frank

middle: V

last: de Paoli

first: Hanne

middle: C

last: Bertram

first: Martin

middle: R

last: Jakobsen

first: Thomas

last: Poulsen

middle: K

last: Holm


title: Nrf2 regulated genes in A549 cells

first: Yonglun

last: Luo


title: REVERSIR platform for the tailored control of GalNAc-siRNA conjugate pharmacology

city: Cambridge

first: Joseph

last: Barry

state: MA

address: 300 Third Street #3

institute: Alnylam Pharmaceuticals

summary:
Primary mouse hepatocytes were treated with 1 nM TTR-siRNA and 10 nM scrambled or TTR-REVERSIR by in vitro free uptake for 24 h. RNA extracted with the Purelink RNA kit (ThermoFisher) was used for cDNA library preparation with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the NextSeq500 desktop sequencer (Illumina), all according to manufacturers’ instructions. Raw RNA-seq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using fastq-mcf. Filtered reads were aligned to the Mus musculus genome (NCBIM37) using STAR with default parameters. Uniquely aligned reads were counted by featureCounts. Differential gene expression (DEG) analysis was performed by the R package DESeq2.
Here, we report the rapid and potent reversal of GalNAc-siRNA-mediated RNAi activity in vivo with REVERSIR technology, which is based on short, synthetic high-affinity oligonucleotide complementary to the siRNA guide strand. The modular, sequence-specific nature of the REVERSIR platform has the potential to enhance the therapeutic profile of any long-acting GalNAc-siRNA conjugate to enable fine-tuned control of RNAi pharmacology

overall_design: RNASeq analysis of REVERSIR in primary mouse hepatocytes

first: Ivan

last: Zlatev

first: Adam

last: Castoreno

first: Christopher

last: Brown

first: June

last: Qin

first: Scott

last: Waldron

first: Mark

last: Schlegel

first: Rohan

last: Degaonkar

first: Svetlana

last: Shulga-Morskaya

first: Huilei

last: Xu

first: Swati

last: Gupta

first: Shigeo

last: Matsuda

first: Akin

last: Akinc

first: Kallanthottathil

middle: G

last: Rajeev

first: Muthiah

last: Manoharan

middle: A

last: Maier

first: Vasant

last: Jadhav


title: Transcriptomic analysis of control lung alveolar type 2 cells and constitutively active-BMPR1a overexpressing alveolar type 2 cells

city: Durham

first: Yoshihiko

last: Kobayashi

state: NC

address: 307 Research Dr

department: Cell Biology

institute: Duke University

summary: The Bone Morphogenetic Protein (BMP) signaling pathway functions in lung development and the regeneration of adult tracheal epithelium from basal stem cells. Here, we explore its role in the alveolar region, where Type 2 epithelial cells (AT2s) and Pdgfrα+ Type 2 - associated stromal cells (TASCs) are components of the stem cell niche. We utilize both organoid assays and in vivo alveolar regrowth after pneumonectomy (PNX) - a process requiring proliferation of AT2s and differentiation into Type 1 cells (AT1). BMP signaling is normally active in AT2s and TASCs, transiently declines post-PNX, and is restored during differentiation of AT2s to AT1s. In organoids, BMP4 inhibits AT2 proliferation, while antagonists promote AT2 self-renewal at the expense of differentiation. Gain and loss-of function genetic manipulation reveals that loss of BMP signaling in AT2s after PNX allows their self-renewal but significantly reduces differentiation into AT1s; conversely, increased BMP signaling promotes AT1 formation. The transcriptome analysis of control AT2s and AT2s overexpressing constitutively active-Bmpr1a reveals that 119 genes are upregulated more than two-fold following enhancing BMP signaling. Of these genes, 10 % are normally preferentially expressed in AT1 cells. Of the 25 genes downregulated more than two-fold, 28 % are preferentially expressed in AT2s.

overall_design: mRNA was isolated from FACS-sorted cells and sequenced using Illumina HiSeq X

first: Mei-I

last: Chung

first: Brigid

last: Hogan


title: Genome wide enrichment/association of Pygo2 with chromatin in unsynchronized HelaS3 cells

city: St. John's

first: Phillip

last: Andrews

state: NL

address: HSC3307, 300 Prince Philip Dr.

department: Biomedical Sciences

country: Canada

institute: Memorial University

summary: We report that the Pygopus (Pygo) 2 chromatin effector is associated with the regulatory elements and utilized for the expression of a set of MYC-dependent genes necessary for cell division. Global chromatin association studies revealed enrichment of Pygo2 at transcriptional start sites in which approximately 60% of Pygo2-associated genes were shared with MYC oncogene. Pygo2 and MYC were co-enriched at promoters/enhancers along with the histone mark H3K27Ac at critical MYC target genes involved in mitotic cell division. Pygo2 interacted with MYC and was required for MYC-dependent transcription and cell growth in a MYC-driven prostate tumor cell model. Our evidence supports a role for Pygo2 in a MYC-dependent transcriptional program that directs formation of the mitotic apparatus.

overall_design: Replicates of Pygo2 ChIP-seq data along with the corresponding input controls

first: P

first: K

last: Kao


title: microRNA profile expression analysis of Human Induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs)

city: Stanford

first: Marta

last: Diez Cunado

state: Ca

address: 300 pasteur dr rd

institute: Stanford University

type: Non-coding RNA profiling by high throughput sequencing

summary: Analysis of the microRNA profile exression in hiPSC-CMs. Results provide important information of the miRNAs expressed in hiPSC-CMs under control conditions.

overall_design: miRNAs obtained from two replicates of hiPSC-CMs purchased from Cellular Dynamics (CDI), Inc. after 14 days of culture and sequencing using Illumina NextSeq500 plattform.

middle: D

last: Cunado

last: Mercola


title: PNPase knockout results in mtDNA loss and an altered metabolic gene expression program

city: Boston

first: Fasih

middle: Mubtasim

last: Ahsan

address: 185 Cambridge Street, CPZN 6500

department: Center for Genomic Medicine

institute: Massachusetts General Hospital

summary: Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and several human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. PKO resulted in loss of mitochondrial DNA (mtDNA) and a transcriptional profile similar to rho0 mtDNA deleted cells, with perturbations in cholesterol, lipid, and secondary alcohol metabolic pathways.  PKO cells also showed growth and cell cycle profiles similar to rho0 cells. PKO in mouse inner ear hair cells cause progressive hearing loss that parallel human familial hearing loss previously linked to mutations in PNPase. Combined, our data suggest that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.

overall_design: Biological triplicate (n = 3) each of Pnpt1 fl/fl mouse embryonic fibroblast (MEF) (TM6), ditercalinium dichloride dihydrochloride treated Pnpt1 fl/fl mtDNA-deficient MEF (rho0), and Pnpt1 fl/fl Adenoviral-Cre treated PNPase KO MEF (PKO) cultures (12 samples total).

first: Michael

last: Teitell


title: Kir4.1-dependent astrocyte-fast motor neuron interactions are required for peak strength

city: San Francisco

first: Kevin

middle: William

last: Kelley

state: CA

address: 35 Medical Center Way

institute: UCSF

summary: Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward rectifying K+ channel Kir4.1 (aka, Kcnj10) around MNs in a VGLUT1-dependent manner. Loss-of astrocyte-encoded Kir4.1 selectively altered FαMN size, function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell-autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation uncouples symptoms of muscle weakness from MN cell death in ALS.

overall_design: 8 samples total consisting of 4 biological replicates per group (Aldh1l1-cre:Kir4.1-floxed/floxed vs cre-negative) of flow sorted Aldh1l1-GFP+ astrocytes from spinal cord at P12-P14

middle: W

first: Lucile

last: Ben Haim

first: David

last: Rowitch


title: Novel Transcriptional Activity and Extensive Allelic Imbalance in the Human MHC Region

city: Ann Arbor

first: Richard

last: McEachin

state: United States

address: 2800 Plymouth Road

department: DCM&B

institute: University of Michigan

summary: The MHC region encodes HLA genes and is the most complex region in the human genome. The extensive polymorphic nature of the HLA hinders accurate localization and functional assessment of disease risk loci within this region. Using targeted capture sequencing and constructing individualized genomes for transcriptome alignment, we identified 908 novel transcripts within the human MHC region. These include 593 novel isoforms of known genes, 137 antisense strand RNAs, 119 novel long intergenic noncoding RNAs, and 5 transcripts of 3 novel putative protein-coding human endogenous retrovirus genes. We revealed allele-dependent expression imbalance involving 88% of all heterozygous transcribed single nucleotide polymorphisms throughout the MHC transcriptome. Among these variants, we show that the genetic variant associated with Behc ̧et’s disease in the HLA-B/MICA region, which tags HLA-B*51, is within novel long intergenic noncoding RNA transcripts that are exclusively expressed from the haplotype with the protective but not the disease risk allele. Further, we showed that the transcriptome within the MHC region can be defined by 14 distinct coexpression clusters, with evidence of coregulation by unique transcription factors in at least 9 of these clusters. Our data suggest a very complex regulatory map of the human MHC, and can help uncover functional consequences of disease risk loci in this region.

overall_design: RNA-Seq in human MHC region

first: Elizabeth

last: Gensterblum-Miller

first: Weisheng

last: Wu

first: Amr

last: Sawalha


title: Transcriptional changes after overexpression of proliferation drivers in human mammary epithelial cells.

city: BOSTON

first: Teresa

last: Davoli

address: 12 Aberdeen Street, 2

institute: BRIGHAM AND WOMEN S Hospital

summary: Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of human cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes tested regulate proliferation, many performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in SCNAs (somatic copy number changes) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue type-specific genetic network architectures underlie SCNA selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive new insights about the genetic network architecture of aneuploidy in tumors. KRTAPs are a class of human genes that promote proliferation in mammary epithelial cells (HMEC), but the mechanism is not understood. We performed RNAseq to study transcriptional changes associated with oeverxepression of KRTAPs and other oncogenes in hTERT-immortalized human mammary epithelial cells. GSEA analysis revealed the top enriched pathways upregulated by KRTAP expression are E2F-mediated regulation of DNA replication, G1-S specific transcription, cell cycle, translation and ribosome. KRTAP-induced mRNA changes are most closely related to those due to CCND1 expression, including induction of E2F1 transcription factor.

overall_design: Analysis of whole transcriptome in HMEC overexpressing different human genes.

first: Laura

last: Sack

first: Eric

last: Wooten

first: Kamila

last: Naxerova


title: Loss of adrenergic nerves in bone marrow drives hematopoietic stem cell niche aging

city: Bronx

first: Paul

last: Frenette

state: NY

address: 1301 Morris Park Avenue , Room 101

department: Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research

institute: Albert Einstein College of Medicine

summary: Aging of hematopoietic stem cells (HSCs) is associated with the decline of their regenerative capacity, and multi-lineage differentiation potential, contributing to development of blood disorders. The bone marrow microenvironment was recently suggested to influence HSC aging, however the underlying mechanisms remain largely unknown. Here, we show that HSC aging critically depends on bone marrow innervation by the sympathetic nervous system (SNS), as premature loss of SNS nerves or adrenoreceptor b3 (ADRb3) signaling in the microenvironment accelerated the appearance of HSC aging phenotypes reminiscent of physiological aging. Strikingly, supplementation of ADRb3 sympathomimetics to old mice significantly rejuvenated in vivo function of old HSCs, suggesting that the preservation or restitution of SNS innervation during aging may hold the potential for novel HSC rejuvenation strategies.

overall_design: mRNA profiles of sorted bone marrow hematopoietic stem cells were generated from saline treated young and old and BRL37344 treated old mice in triplicates by Illumina NextSeq500 sequencing

first: Qiaozhi

last: Wei


title: Direct reprogramming of mouse fibroblasts into functional skeletal muscle progenitors

first: Ori

last: Bar-Nur

address: 185 Cambridge st.

department: Center of Regenerative medicine

institute: Harvard University

summary: Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to Dystrophin-expressing myofibers upon transplantation, a subset of which maintain Pax7 expression in vivo and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that iMPCs can be established from muscle tissue following small molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple differentiated cell types.

overall_design: Examination of the transcriptome of induced myogenic progenitor cells in comparison to satellite cells

first: Konrad

last: Hochedlinger


title: Large-scale expansion of human iPSC-derived skeletal muscle cells for disease modeling and cell-based therapeutic strategies

status: Public on May 03 2018

city: Rotterdam

last: Pijnappel

address: Wytemaweg 80

country: Netherlands

institute: Erasmus MC

summary: Although skeletal muscle cells can be generated from human iPSCs, transgene-free protocols include only limited options for their purification and expansion. In this study we found that FACS-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 x 1011 fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of the acid alpha glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies.

overall_design:
Myogenic progenitors were expanded for ~15 days and harvested either in proliferation conditions or after 4 days of differentiation as described previously (van der Wal et al., 2017b). RNA was extracted using the RNeasy minikit with DNAse treatment (Qiagen, Germantown, MD). Sequencing libraries were prepared using TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, California, USA) according to the manufacturer’s instructions. Libraries were sequenced on a HiSeq2500 sequencer (Illumina, San Diego, California, USA) in rapid-run mode according to the manufacturer’s instructions. Reads 50 base-pairs in length were generated. The RNA-sequencing datasets listed in table S3 were downloaded and aligned with the datasets generated in this study using the ‘new Tuxedo’ pipeline (Pertea et al., 2016).

The processed data file includes the analysis of 30 additonal Samples from other research groups, partly from GEO and partly from other sources such as ENCODE and ENA. The header table linked below lists the origin of the other Samples.

first: Erik

last: van der Wal

first: Pablo

last: Herrero-Hernandez

first: Raymond

last: Wan

first: Mike

last: Broeders

first: Stijn

last: in 't Groen

first: Tom

middle: J

last: van Gestel

first: Wilfred

middle: F

last: van IJcken

middle: H

last: Cheung

first: Ans

middle: T

last: van der Ploeg

first: Gerben

last: Schaaf

first: W

last: Pim Pijnappel


title: Microarray analysis of TFEB-regulated genes in endothelial cells

first: Haocheng

last: Lu

state: Michigan

address: 2800 plymouth road

department: Internal Medicine

summary:
Transcription factro-EB (TFEB) is a master gene for autophagy and lysosome biogenesis
We used microarrays to detail the gene expression in TFEB-overexpressing endothelial cells and identified distinct classes of TFEB-regulated genes.

overall_design: Human umbilical vein endothelial cells (HUVEC) overexpressing control GFP or TFEB were used for RNA extraction and hybridization on Affymetrix microarrays.

first: Yanbo

last: Fan

first: Yuqing

middle: E

last: Chen


title: Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation

city: Bethesda

first: Aleksandra

middle: M

last: Michalowski

state: Maryland

address: 37 Convent Drive

institute: National Cancer Institute

summary: Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared to single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared to single agents. Mice, bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared to single agents. A set of 37 genes cooperatively affected (34 down-regulated; 3 up-regulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes down-regulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared to normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo.

overall_design: Gene expression was profiled from L363 cells treated with either 1nM or 10nM rapamycin, 0.5uM MS-275 or the combination for 48 hours. Three replicates per control and treatment groups were done; one array was disqualifed with QC.

first: Beverly

last: Mock

first: John

last: Simmons

first: Jyoti

last: Patel

first: Ke

last: Zhang


title: Expression data from surface airway epithelial basal cells, ciliated cells, and club cells.

status: Public on Apr 30 2018

city: Iowa City

last: Engelhardt

state: Iowa

address: 1-111L BSB 51 Newton Rd.

department: Anatomy and Cell Biology

institute: University of Iowa

summary:
To identify key genes that define surface airway epithelial (SAE) basal cells, we FACS isolated basal, ciliated, and club cell populations as previously reported (Zhao et al., 2014; PMID: 25043474) and performed microarray analysis on isolated mRNA. For fractionating SAE into basal, club, and ciliated populations, cells were stained with EpCAM-PECy7 (eBiosciences), GSIβ4-FITC (Sigma), SSEA1-Alexa Fluor® 647 (BioLegend), and CD24-PE (BD Pharmingen) for 30 minutes on ice as previously described (Zhao et al., 2014), prior to FACS. Basal cells were considered EpCAM+ and GSIβ4+. Secretory cells were considered EpCAM+ and SSEA1+. Ciliated cells were considered EpCAM+, GSIβ4- and CD24+.
Primary SAE cells were harvested from C57BL/6 mice and sorted into basal, ciliated, and club cell populations for the purpose of identifying enrichment of transcripts specific to each cell type population.

overall_design: Total RNA from basal, ciliated, and club cell populations were isolated and assayed.

first: J

first: T

last: Lynch


title: The effects of Lef-1 overexpression on culture-expanded MECs

summary: The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair—basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

overall_design: Five Lef-1KI samples and four wild type samples were analyzed.

first: Thomas


title: Quantitative Analysis of Follicular Helper T cell Transcriptomes from Wild Type and Apoe-/-

city: Seoul

first: Heeju

last: Ryu

address: 1 Gwanak-ro

country: South Korea

institute: Seoul National University

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goal of this study is to characterize the pathogenic features of follicular helper T cells (TFH cells) generated in atherogenic conditions by NGS-derived transcriptome profiling (RNA-seq).
Methods: TFH cell mRNA profiles of 13-week-old wild type (WT) and Apoe knockout (Apoe-/-) mice were generated by deep sequencing, in duplicate, using Invitrogen mirVana miRNA Isolation Kit (Cat#AM1561). The sequence reads that passed quality filters were analyzed by TopHat (version 2.0.13) followed by Cufflinks (version 2.2.0).
Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (build mm10) and identified 23997 transcripts in the retinas of WT and Apoe–/– mice with approximately 88% mapping rates. At a setting of greater than two-fold expression changes, p value < 0.05, and false discovery rate (FDR) < 0.1, 211 genes were upregulated and 142 genes were downregulated in TFH cells from Apoe–/–mice compared with those from WT mice.
Conclusions: Our study demonstrates detailed transcriptome analysis of TFH cells from WT and Apoe–/–, with biologic replicates, generated by RNA-seq technology.

overall_design: mRNA profiles of follicular T helper cells from wild type and atherogenic mice were generated by deep sequencing, in duplicate, using Illumina Truseq.

first: Chung

last: Yeonseok

first: Ryu

last: Heeju


title: DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library established in haploid human embryonic stem cells (hESCs)

city: Jerusalem

first: Mordecai

last: Peretz

address: The Edmond J. Safra Campus

country: Israel

institute: Hebrew University of Jerusalem

summary: The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs.  We could allude to an intrinsic bias of essentiality across cellular-compartments, uncover two opposing roles for tumor-suppressor genes, and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear-factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss-of-function provides growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.

overall_design: Haploid hESCs were transduced with a lentiviral pooled gRNA library carrying 181,131 gRNAs at a multiplicity of infection of 0.3 to enrich for single integrations/mutations per cell. Transduced haploid hESCs were selected with puromycin and cultured for 30 days. Mutant hESCs were passaged every 4-5 days and DNA samples were collected from the population at multiple time points during the passaging. gRNA sequences were amplified from the genomic DNA and analyzed by high-throughput deep sequencing.

first: Atilgan

last: Yilmaz

first: Nissim

last: Benvenisty


title: Characterization of missense mutations in the tetratricopeptide region of O-GlcNAc transferase found in patients with X-linked intellectual disability

city: Athens

first: Lance

last: Wells

state: GA

address: 315 Riverbend Rd

institute: Complex Carbohydrate Research Center, University of Georgia

summary: Dfferential transcriptomics analysis of RUES-1 human embryonic stem cells edited using Crispr/Cas9 to contain four mutations in OGT found in certain patients with X-linked intellectual disability reveals changes in the gene expression profile associated with ectoderm and mesoderm development in all four mutant cell lines compared to the wild type control.

overall_design: The steady state global gene expression profiles of four mutant cell lines are compared with that of a control wild type cell line using Illumina mRNA-sequencing.

first: Nithya

last: Selvan

first: Nripesh

last: Prasad

first: Shawn

last: Levy


title: Setd2 regulates quiescence and differentiation of adult hematopoietic stem cells by restricting RNA polymerase II elongation

city: Cincinnati

first: Gang

last: Huang

state: Ohio

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: SET domain containing 2 (Setd2), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we described an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelet accompanied with hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation, because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit of quiescence and commit to differentiation, which lead to hematopoietic stem cells exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes including Myc. Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of HSCs through restricting the NSDs/SEC mediated RNA Polymerase II elongation.

overall_design: mRNA-seq of wild-type Setd2 (Setd2f/f) and Setd2 knock-out (Setd2D/D) LSK cells were performed in the study.

first: Yile

last: Zhou


title: Epigenetic profiling of sorted tumor cells from implanted T cell low and T cell high mouse pancreatic tumors

city: New York

first: Jinyang

last: Li

state: New York

address: 1230 York Ave

institute: The Rockefeller University

summary:
Purpose: Use ATAC-seq strategy to characterize the differentially openned chromatin region in T cell low and high mouse pancreatic tumors in order to identify factors contributing to the differential abundance of tumor infiltrating T cells.
Methods: Subcutaneously implanted T cell low and high pancreatic tumors were utilized for the preparation of ATAC-seq libraries from sorted tumor cells.

overall_design: Examination of differentially openned chromatin region between two biological groups (T cell low v.s. T cell high tumors).

first: Ben

middle: Z

last: Stanger

first: Robert

last: Vonderheide

first: Katelyn

last: Byrne


title: Transcriptome analysis of human embryonic stem cells (hESCs) and human fibroblasts upon rapamycin

summary: Rapamycin inhibits mTORC1 complex in a variety of cells. We demonstrated that hESCs are more sensitive to rapamycin treatment as compared to somatic cells (BJ Fibroblasts) and also certain cancer cells (KBM7 cells). To study the molecular mechanisms of this disparity, we performed a transcriptome analysis (RNAseq) of rapamycin-treated hESCs and human fibroblasts for two time points (2 and 4 days of treatment).

overall_design: Human embryonic stem cells (hESCs) and human fibroblasts were treated with rapamycin for two and four days. For untreated controls and each treatment condition three biological replicates were analyzed.


title: Long noncoding RNA Hoxb3os is dysregulated in autosomal dominant polycystic kidney disease and regulates mTOR signaling

city: Minneapolis

first: Micah

last: Gearhart

state: MN

address: 6-160 Jackson Hall, 321 Church St.

department: Genetics, Cell Biology and Development

institute: University of Minnesota

summary: Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating disease that is characterized by the accumulation of numerous fluid-filled cysts in the kidney. ADPKD is primarily caused by mutations in two genes, PKD1 and PKD2. Long noncoding RNAs (lncRNA) – defined by a length >200 nucleotides and absence of a long open reading frame – have recently emerged as epigenetic regulators of development and disease; however, their involvement in PKD has not been explored previously. Here, we performed deep RNA sequencing to identify lncRNAs that are dysregulated in two orthologous mouse models of ADPKD (kidney-specific Pkd1 and Pkd2 mutant mice). We identified a kidney- specific, evolutionarily-conserved lncRNA called Hoxb3os that was downregulated in cystic kidneys from Pkd1 and Pkd2 mutant mice. The human ortholog HOXB3-AS1 was downregulated in cystic kidneys from ADPKD patients. Hoxb3os was highly expressed in renal tubules in adult wild- type mice, whereas its expression was lost in the cyst epithelium of mutant mice. To investigate the function of Hoxb3os, we utilized CRISR/Cas9 to knockout its expression in mIMCD3 cells. Deletion of Hoxb3os resulted in increased phosphorylation of mTOR and its downstream targets, including p70 S6 kinase, ribosomal protein S6, and the translation repressor 4E-BP1. Consistent with activation of mTORC1 signaling, Hoxb3os mutant cells displayed increased mitochondrial respiration. The Hoxb3os mutant phenotype was partially rescued upon re- expression of Hoxb3os in knockout cells. These findings identify Hoxb3os as a novel lncRNA that is downregulated in ADPKD and regulates mTOR signaling and mitochondrial respiration.

overall_design: Expression profiling by sequencing in primary kidney and cultured cells.

first: K

last: Aboudehen

first: M

first: P

last: Igarashi


title: Changes in corticotrope gene expression upon increased expression of peptidylglycine a-amidating monooxygenase, an evolutionarily conserved enzyme

city: Farmington

first: Richard

last: Mains

address: 263 Farmington Ave, Neuroscience Dept., Neuroscience Dept.

department: Neuroscience

institute: University of Connecticut Health Center

summary: Throughout evolution, secretion has played an essential role in the ability of organisms and single cells to survive in the face of a changing environment. PAM (peptidylglycine a-amidating monooxygenase) is an integral membrane monooxygenase first identified for its role in the biosynthesis of neuroendocrine peptides released by the regulated secretory pathway.  By screening for transcripts whose expression in a corticotrope tumor cell line responded to changes in PAM levels, we sought to identify genes not previously known to play a role in secretion.  We identified transcription factors, peptidyl prolyl isomerases, endosomal/lysosomal  proteins and proteins involved in tissue-specific responses to glucose and amino acid availability that had not previously been recognized as participants in secretion.

overall_design: Examination of mRNA profiles in corticotropes expressing basal or induced levels of PAM, followed by quantitative PCR and Western blot analyses.

first: Bruce

last: Rheaume


title: Trans-differentiation of human adult peripheral blood T cells into neurons

status: Public on Apr 28 2018

first: Cheen Euong

last: Ang

address: 265 Campus Drive, G3145

institute: Stanford University

summary: Examining the transcriptomic changes during transdifferentiation of peripheral blood mononuclear cells to induced neuronal cells.

overall_design: There are three different populations: PBMC (2 biological replicates, starting population), PSA-NCAM+GFP+ (2 biological replicates, induced neuronal cells) and PSA-NCAM+GFP- (2 biological replicates, induced neuronal cells).

first: Cheen

first: Koji

last: Tanabe


title: Analysis of a case of esophageal NEC at transcriptome level

city: Shantou

first: Qian

last: Yang

state: Guangdong

address: Xinling 22 Road

institute: Medical College of Shantou University

summary: RNA-seq of a paired esophageal NEC sample and the corresponding normal esophageal sample from the same individual.

first: En-Min

first: Li-Yan

last: Xu


title: Epigenomics analysis of an esophageal NEC case

summary: We detected DNA methylation of an esophageal NEC sample and the corresponding normal sample to identify the aberrant DNA methylation status in this rare disease.

overall_design: Bisulphite converted DNA from the normal and tumor samples from the same individual were hybridised to the Illumina Infinium 450k Human Methylation Beadchip

first: Jian-Zhong

last: He


title: DAOY-Notch1/Notch2 knockout: transcriptome comparison

city: Solna

first: Urban

last: Lendahl

address: Berzelius väg 35

department: CMB

country: Sweden

institute: Karolinska Institutet

summary: Hyperactivation of Notch signaling and the cellular hypoxic response are frequently observed in cancers, with increasing reports of connections to tumor initiation and progression. The two signaling mechanisms are known to intersect, but while it is well established that hypoxia regulates Notch signaling, less is known about whether Notch can regulate the cellular hypoxic response. We now report that Notch signaling specifically controls expression of HIF2a, a key mediator of the cellular hypoxic response. Transcriptional upregulation of HIF2a by Notch under normoxic conditions leads to elevated HIF2a protein levels in primary breast cancer cells as well as in human breast cancer, medulloblastoma and renal cell carcinoma cell lines. The elevated level of HIF2 protein was in certain tumor cell types accompanied by down-regulation of HIF1a protein levels, indicating that high Notch signaling may drive a HIF1a-to-HIF2a switch. At the transcriptome level, the presence of HIF2a was required for approximately 21% of all Notch-induced genes: among the 1062 genes that were upregulated by Notch in medulloblastoma cells during normoxia, upregulation was abrogated in 227 genes when HIF2a expression was knocked down by HIF2a siRNA. In conclusion, our data show that Notch signaling affects the hypoxic response via regulation of HIF2a, which may be important for future cancer therapies.

overall_design: Transcriptome analyis of DAOY NOTCH1 or NOTCH2 deficient cells cultured on Jagged1-ligand coated plates to activate Notch signalling, in combination  with the pan-Notch inhibitor DAPT for 12h to block Notch signalling.


title: Next-generation sequencing analysis of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts (MEFs)

status: Public on Apr 27 2018

city: Abu Dhabi

first: Piergiorgio

last: Percipalle

state: Abu Dhabi

address: Saadiyat Campus, Abu Dhabi

department: Biology Department, Science Division

country: United Arab Emirates

institute: New York University Abu Dhabi

summary:
Purpose: The goals of this study are to use NGS to perform transcriptome profiling (RNA-seq) to find the difference of the transcriptomes of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts.
Methods: MEFs were reprogramed into neurons by small chemical cocktails. Small chemical molecules were dissolved and diluted in DMSO and used at the following final concentrations: ISX9: 20 mM; Forskolin: 50 mM; CHIR99021: 20 mM; and I-BET151: 2 mM. In addition to the small molecules, the neuron induction medium (Neurobasal Medium) contains the following supplements: N2 (1X) and B27 (2X) supplements, GlutaMAX (1X), penicillin-streptomycin (100 µg/ml), bFGF (20 ng/ml), 100 µM cAMP, Non-essential Amino Acid (1X) and Trace element B (1X). MEFs were seeded to Matrigel-coated plate (1:30 dilution in pre-cold PBS and coat overnight at 4 oC, at a density of 200,000 cells per well in 6-well plate and 10,000 cells/well in 96 well plate. The MEFs were cultured in DMEM until confluent. When the cells are confluent, the DMEM was replaced with neuron induction medium with 4 small molecules. The induction medium was refreshed every two days for the first week and every 3 days for the remaining induction period until day 20. 3 biological replicates of total RNA were used for RNA-seq library preparation.
Results: Using an optimized data analysis workflow, we identified neuronal gene programs induced during direct reprograming. The induction of neuronal programs were impaired in neurons from Actb-/- mouse embryonic fibroblasts.
Conclusions: Our study shows that neuronal program induction was impaired in chemically induced neurons from Actb-/- mouse embryonic fibroblasts.

overall_design: mRNA profiles of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts were generated by deep sequencing, in 3 biological replicates.

first: Xin

last: Xie

first: Nizar

last: Drou


title: De novo lipogenesis represents a therapeutic target in Kras mutant NSCLC

city: Worcester

first: George

last: Acquaah-Mensah

address: 19 Foster Street

institute: MASS COLLEGE OF PHARM HEALTH SCIENCES

summary:
We examine the potential of Kras as a metabolic target in lung cancer using the KrasLSL-G12D lung cancer model. We demonstrate that mutant Kras drives a lipogenic gene expression program, and that fatty acid synthesis is important in Kras-induced tumorigenesis.
Compare gene expression changes between mutant Kras (G12D) driven lung tumors and normal lung samples to identify pathways selectively altered in lung tumors.

overall_design: To identify the pathways selectively modulated by oncogenic Kras (G12D) signaling in lung tumors, we used the transgenic Kras LSL-G12D model of lung cancer. By crossing these mice with another transgenic mice (CC10_Cre)  expressing Cre recombinase selectively in Clara cells of the lung, we generated Kras LSL-G12D CC10-Cre mice which selectively express mutant kras in clara cells. These mice develop lung adenocarcinoma and have a median survival of 80 days.

first: Anju

last: Singh

first: Shyam

last: Biswal


title: Expression and methylation profiling of esophageal NEC

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

overall_design: Refer to individual Series


title: Non-clinical and clinical pharmacology of the potent and selective RARγ agonist trifarotene

city: Sophia Antipolis

first: CORINNE

last: MENIGOT

address: 2400 route des colles

country: France

institute: GALDERMA R & D

summary: Background: First- and third-generation retinoids are the main treatment in acne. Even though efficacious, they lack full selectivity for RARγ expressed in the epidermis and infundibulum.   Objectives: To characterize the in vitro metabolism and the pharmacology of the novel retinoid trifarotene.  Methods: In vitro assays determined efficacy, potency and selectivity on RARs, as well as the activity on the expression of retinoid target genes in human keratinocytes and ex vivo cultured skin. In vivo studies investigated topical comedolytic, anti-inflammatory and depigmenting properties. The trifarotene-induced gene expression profile was investigated in non-lesional skin of acne patients and compared to ex vivo and in vivo models. Finally, the metabolic stability in human keratinocytes and hepatic microsomes was established.  Results: Trifarotene is a selective RARγ agonist with >20-fold selectivity over RAR and RARβ. Trifarotene is active and stable in keratinocytes but rapidly metabolized by human hepatic microsomes, predicting improved safety. In vivo, trifarotene 0.01% applied topically is highly comedolytic and has antiinflammatory and antipigmenting properties. Gene expression studies indicated potent activation of known retinoid-modulated processes (epidermal differentiation, proliferation, stress response, RA metabolism) and novel pathways (proteolysis, transport/skin hydration, cell adhesion) in ex vivo and in vivo models, as well as in human skin after four weeks of topical application of trifarotene 0.005% cream.   Conclusion: Based on its RARγ selectivity, rapid degradation in human hepatic microsomes and pharmacological properties including potent modulation of epidermal processes, topical treatment with trifarotene is expected to provide strong efficacy combined with a favourable safety profile in acne and ichthyotic disorders.

overall_design: Total of 16 chips. 9 patients : 2 biospies per patient: 1 in non-inflammatory skin of acne patient after vehicle treatment (J27) (Back), 1 in non-inflammatory skin of acne patient after after CD 5789 treatment (J27) (Back)

first: Johannes

last: Voegel


title: Setd2-dependent H3K36me3 is required for Acrbp1 and protamine transcriptions and spermiogenesis in mice

city: Shanghai

first: Fei

last: Lan

address: No131 Dongan Road, Xuhui District.

institute: Institute of Biochemical Science

summary: Spermatogenesis is precisely cotrolled by complex gene expression programs and involves epigenetic reprogramming including histone modification and DNA methylation. Setd2 catalyzes the trimethylation of histone H3 Lys36 (H3K36me3) and plays key roles in embryonic stem cell differentiation and somatic cell development; however, its role in male germ cell development remains elusive. Here we demonstrate an essential role of Setd2 for spermiogenesis. We show that targeted knockout of Setd2 in germ cells causes aberrant spermiogenesis with acrosomal malformation before step 8 round spermatid stage, resulting in complete male infertile. Furthermore, we show a complete loss of H3K36me3 and a significant altered gene expression profile, including Acrbp1 and protamines, caused by Setd2 deficiency. Our findings reveal a previously underappreciated role of Setd2-dependent H3K36me3 for spermiogenesis and improved the understanding of epigenetic disorders underlying male infertility.

overall_design: We examined H3K36me3 and transcriptomes in in PD and RS cells upon Setd2 loss.

first: Bowen

last: Rong

first: Li

last: Li

first: Minghan

last: Tong

first: Ruitu

last: Lyu


title: A single-cell transcriptome atlas of mouse glomerulus

city: Berlin

first: Nikos

last: Karaiskos

address: Hannoversche Str. 28

country: Germany

institute: Max Delbrück Center for Molecular Medicine

summary: Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. As yet, it remains unknown to what extent cellular heterogeneity exists within healthy glomerular cell populations. Here, we used nanodroplet-based, highly parallel transcriptional profiling to characterize the cellular content of purified wildtype mouse glomeruli. Unsupervised clustering of 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells, which can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry stainings obtained from the Human Protein Atlas. Subclustering of glomerular endothelial cells revealed a subset of activated endothelium, expressing marker genes related to endothelial proliferation. Additionally, the podocyte population could be divided in three different subclusters. In conclusion, our study comprehensively characterizes gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

overall_design: Single cell transcriptomics of Mouse Kidney Glomeruli using Drop-seq

first: Mahdieh

last: Rahmatollahi

first: Christine

last: Kocks

first: Martin

last: Kann

first: Roman-Ulrich

last: Mueller


title: Genome-wide maps of Nrf2 and Pol2 binding in A549 cells

city: aarhus

first: Yonglun

last: Luo

address: Wilhelm Meyers Allé 4

institute: Aarhus University

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

overall_design: Nrf2 and Pol2 binding peaks of A549 cells were generated by ChIP deep sequencing using Active Motif (Carlsbad, California, USA), Illumina’s NextSeq 500.

first: David

last: Olagnier

first: Aske

last: Brandtoft

first: Camilla

last: Gunderstofte

first: Nikolaj

middle: L

last: Villadsen

first: Christian

last: Krapp

first: Anne

last: Thielke

first: Anders

last: Laustsen

first: Suraj

last: Peri

first: Anne-Louise

last: Hansen

first: Lene

last: Bonefeld

first: Jacob

last: Thyrsted

first: Lin

last: Lin

first: Victor

last: Bruun

first: Marie

middle: B

last: Iversen

first: Virginia

last: Artegoitia

first: Rongtuan

first: Siddharth

last: Balachandran

first: Mette

last: Nyegaard

first: Bernadette

last: Marrero

first: Raphaela

last: Goldbach-Mansky

first: Luke

last: O’Neill

first: Frank

middle: V

last: de Paoli

first: Hanne

last: Bertram

last: Jakobsen

first: Thomas

last: Poulsen

last: Holm


title: Genetic dissection of the α-globin super-enhancer in vivo [RNA-seq]

city: Oxford

first: Jelena

last: Telenius

address: MRC Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington

department: Weatherall Institute of Molecular Medicine (WIMM)

country: United Kingdom

institute: Oxford University

summary: Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

overall_design: Mouse fetal liver erythroid RNA-seq. The RNA of the erythroid cells was metabolically labelled using 4-thiourdine nucleotide analogue supplementation of viable cells in culture. RNA transcripts that incorporated the analogue and hence were synthesised during this period of exposure, were then isolated from the pre-exiting bulk RNA by the addition of a biotin moiety and pull down.

first: Deborah

last: Hay

first: Jim

middle: R

last: Hughes

first: Douglas

last: Higgs


title: Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

status: Public on Apr 26 2018

city: Ithaca

first: Iwijn

last: De Vlaminck

address: 301 Weill Hall

department: Meinig School of Biomedical Engineering

institute: Cornell University

summary: We developed a tunable ligation technology to alter beads used for to capture mRNA for single-cell RNA sequencing (e.g. Drop-seq); we call this technique droplet assisted RNA targeting by single cell sequencing, or DART-seq. In the study, we applied the new technology to capture viral genome transcripts of recombinant type 3 Dearing orthoreovirus infecting murine L929 cells. In addition to capture of targeted viral gene transcripts, we were also able to simultaneosly pull-down polyadenylated mRNA from the L929 cells to observe host-pathogen interactions. We also applied the technology to efficiently measure natively paired heavy and light chain amplicons from B lymphocyte cells in a collection of human peripheral blood mononuclear cells.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Commercial Drop-seq beads (Chemgene) were altered by a tunable ligation chemistry to capture targets of interest while maintaining the ability to capture polyadenylated mRNAs. Altered or normal beads were resuspended in lysis buffer and were coencapsulated with single cells (either infected murine L929 cells or, in another experiment, human PBMCs) using a microfluidic device (as per standard Drop-seq protocol ; Macasko et al., 2015). cDNA was created from the captured RNA targets and amplified. Nextera library preparation was used to tagment and index the cDNA for next generation sequencing.

first: Philip

last: Burnham


title: JunB defines functional and structural integrity of the epidermo-pilosebaceous unit in the skin

city: Ulm

first: Pallab

last: Maity

address: Meyerhofstrasse/James Franck Ring

department: Dermatology

institute: University of Ulm

summary: Here we identified JunB, a member of the AP-1 transcription factor family, regulated epidermal stem cells and sebaceous glands through balancing proliferation and differentiation of progenitors and by suppressing lineage infidelity.

overall_design: Transcriptome profiling of JunB deficient keratinocytes and post-wounded skin

first: Karin

last: Scharffetter-Kochanek


title: Postprandial FGF19 signaling-induced phosphorylation of FXR by Src modulates its activity in bile acid homeostasis

city: Urbana

first: Young-Chae

last: Kim

state: IL

address: 394 Burrill Hall  407 S. Goodwin Ave

department: MIP

institute: University of Illinois at Urbana-Champaign

summary: Farnesoid-X-Receptor (FXR) plays a central role in maintaining bile acid (BA) homeostasis by transcriptional control of numerous enterohepatic genes, including intestinal FGF19, a hormone that strongly represses hepatic BA synthesis. How activation of the FGF19 receptor at the membrane is transmitted to the nucleus for transcriptional regulation of BA levels and whether FGF19 signaling posttranslationally modulates function of FXR remain largely unknown. Here we show that FXR is phosphorylated at Y67 by non-receptor tyrosine kinase, Src, in response to postprandial FGF19, which is critical for its nuclear localization and transcriptional regulation of BA levels. Liver-specific expression of phospho-defective Y67F-FXR or Src-downregulation in mice result in impaired homeostatic responses to acute BA feeding, and exacerbate cholestatic pathologies upon drug-induced hepatobiliary insults. Also, the hepatic FGF19-Src-FXR pathway is defective in primary biliary cirrhosis patients. This study identifies Src-mediated FXR phosphorylation as a potential therapeutic target and biomarker for BA-related enterohepatic diseases.

overall_design: Liver mRNA profiles were generated by deep sequencing, were profiled in mouse liver in which FXR-floxed mice were co-infected with AAV-TBG-Cre and either AAV-TBG expressing FXR-WT or Y67F-FXR for 4 weeks

first: Sangwon

last: Byun

first: Dong-Hyun

first: Daniel

last: Ryerson

first: Hao

last: Sun

first: Bo

last: Kong

first: Peter

last: Yau

first: Grace

last: Guo

first: H

last: Xu

first: Byron

last: Kemper

first: Jongsook

middle: K


title: RNAi mediates allele-specific epigenetic inheritance of heterochromatin [ncRNA-seq]

city: San Francisco

first: Ruby

last: Yu

address: 513 Parnassus Ave

institute: UCSF

summary: We tested the ability of trans siRNAs to establish a silent allele of endogenous ade6+ or an ade6+ transgene inserted within euchromatic loci. We found that a silent ade6-OFF epi-allele that grows red on low adenine medium could be established in several mutant backgrounds (mlo3∆, leo1∆) in the presence of ectopic siRNAs. Furthermore, we found that the silent ade6-OFF epi-allele could be meiotically inherited to wildtype progeny lacking the establishing mutation or ectopic siRNAs. Here, we sequenced genome-wide small RNAs and found widespread generation of secondary siRNAs at genes surrounding the ade6-OFF locus in cells expressing the ectopic siRNAs source cen::ade6. In wildtype meiotic progeny lacking the driver cen::ade6 siRNAs, we observed establishment of a local siRNA producing locus at genes adjacent to the endogenous ade6-OFF  or at a kanMX-ade6-OFF transgene inserted within euchromatic loci. In contrast, spreading of secondary siRNAs was limited or nonexistent in ade6-ON cells. Finally, we found that spreading of secondary siRNAs at the endogenous ade6 locus was  limited when an ade6 hairpin construct served as the ectopic siRNA driver locus instead of cen::ade6.

overall_design: Non-coding RNA profiling by high throughput sequencing

first: Xiaoyi

last: Wang

first: Danesh

last: Moazed


title: Interrogating the functions of PRDM9 domains in meiosis

city: Bethesda

first: Galina

last: Petukhova

address: 4301 Jones Bridge Rd

department: Biochemistry

institute: USUHS

summary: Homologous recombination is required for proper segregation of homologous chromosomes during meiosis. It predominantly occurs at recombination hotspots that are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains three domains which are typically different families of proteins involved in regulation of transcription in other proteins, yet, the role of PRDM9 in gene expression control has not been evaluated. Here we analyze the germline transcriptome of the  Prdm9-/- male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. We further explore the role of PRDM9 in meiosis by analyzing the activity of PRDM9 lacking effect of the KRAB, SSXRD and post-SET zinc finger domains deletion mutants in a cell expression system and the KRAB domain deletion in mice. We found that although the post-SET zinc finger and the KRAB domains are not essential for the methyltransferase activity of PRDM9 in a cell culture expression systemin vitro, mice lacking the KRAB domain mutant mice  domain show only residual PRDM9 methyltransferase activity and undergo meiotic arrest. In aggregate, our data indicate that these domains that are classically involved in gene regulation do not serve that role in PRDM9, but instead are involved in PRDM9 function has likely diverged from the typical function of a transcription factor and adapted the new role in setting the proper chromatin environment for initiation and completion of homologous recombination.

overall_design:
Testes mRNA-seq, miRNA-seq, Chip-seq were generated using next generation sequencing.

* The "GSE104850_RAW.tar" archive includes the processed data files for Samples GSM3053281-GSM3053299. *

first: Sarah

last: Thibault-Sennett

first: Qi

first: Fatima

last: Smagulova

first: Jeff

last: Cloutier

first: Kevin

last: Brick

first: Dan

last: Camerini-Otero


title: RNA-seq of CD33 KO and control HSPCs

city: Saint Louis

first: Miriam

last: Kim

state: Missouri

address: 660 S. Euclid Ave

institute: Washington University in St Louis

summary: Gene expression profile of in vitro differentiated control and CD33 KO CD34+ cells (with 70-85% CD33 KO) were analyzed by RNA-seq to exclude any major impact of CD33 loss on downstream gene expression

overall_design: Primary CD34+ cells were treated with CRISPR/Cas9 to disrupt the CD33 gene and grown in culture for 5-7 days prior to analysis; mRNA profile was compared to control cells from the same donor that were also treated with Cas9 and a control gRNA; 5 different donors were evaluated (CD33 KO/control for each = total 10 samples)

first: Saar

last: Gill


title: Expression levels of genes of NKG2C+ NK cells after in vitro treatment

first: Pawel

last: Durek

address: Charitéplatz 1

institute: Deutsches Rheuma-Forschungszentrum

summary: RNA-Seq and comparison of gene expression levels was performed of FACS-sorted viable CD56+ NKG2C+ NK cells after in vitro treatment with peptide-pulsed RMA-S/HLA-E/LFA-3 target cells in the presence of or absence of IL-12 and IL-18

overall_design: NK cells of HCMV-seronegative healthy individiuals were FACS-sorted for viable CD3- CD56dim cells. CD56dim NK cells were co-cultured for 7 days with RMA-S/HLA-E/LFA-3 target cells either pulsed with VMAPQSLLL or VMAPRTLFL peptide in the presence of 10 ng/mL IL-15. 10 ng/mL IL-12 and 100 IL-18 were either present or not during the initial 20 h of culture.

first: Quirin

last: Hammer

first: Timo

last: Rückert

first: Eva

last: Borst

first: Josefine

last: Dunst

first: Andre

last: Haubner

first: Gilles

last: Gasparoni

first: Frederik

last: Heinrich

first: Marina

last: Babic

first: Adriana

last: Tomic

first: Gabriella

last: Pietra

first: Mikalai

last: Nienen

first: Igor

last: Blau

first: Jörg

last: Hofmann

first: Il-Kang

first: Immo

last: Prinz

first: Christian

last: Könecke

first: Philipp

last: Hemmati

first: Nina

last: Babel

first: Renate

last: Arnold

first: Jörn

last: Walter

last: Thurley

first: Mir-Farzin

last: Mashreghi

first: Martin

last: Messerle

first: Chiara

last: Romagnani


title: Effect of NORAD shRNA on A549 cells treated with TGF-beta

city: Tokyo

first: Daizo

last: Koinuma

address: Hongo 7-3-1, Bunkyo-ku

department: Pathology

country: Japan

institute: University of Tokyo

summary: We evaluated the effect of NORAD (also known as LINC00657 or LOC647979) shRNA on TGF-beta induced changes in the gene expression in A549 cells by RNA-seq.

overall_design: mRNA expression was determined in a lung adenocarcinoma cancer cell line A549 infected with NORAD shRNA-expressing lentiviral vector and treated with TGF-beta.

first: Natsumi

last: Kawasaki

first: Toshiki

last: Miwa

first: Satoshi

last: Hokari

first: Kohei

last: Miyazono


title: RNAi mediates allele-specific epigenetic inheritance of heterochromatin [ChIP-seq]

first: Ruby

last: Yu

summary: We tested the ability of trans siRNAs to establish a silent allele of endogenous ade6+ or an ade6+ transgene inserted within euchromatic loci. We found that a silent ade6-OFF epi-allele that grows red on low adenine medium could be established in several mutant backgrounds (mlo3∆, leo1∆) in the presence of ectopic siRNAs. Furthermore, we found that the silent ade6-OFF epi-allele could be meiotically inherited to wildtype progeny lacking the establishing mutation or ectopic siRNAs. Here, we sequenced genome-wide DNA associated with conserved heterochromatin markers and found that silencing of the endogenous ade6 locus correlates with increased spreading of H3K9me2 and H3K9me3. Additionally, heterochromatin domains established with cen::ade6 as the ectopic siRNA-producing locus were associated with significantly more spreading of H3K9me2 and H3K9me3 than those established using an ade6 hairpin as the driver siRNA-producing locus.

overall_design: Examination of H3K9me2 or H3K9me3

first: Xiaoyi

last: Wang

first: Danesh

last: Moazed


title: RNAi mediates allele-specific epigenetic inheritance of heterochromatin


title: Allele-specific control of replication timing and genome organization during development

city: Tallahassee

first: David

last: Gilbert

state: FL

address: 319 Stadium Drive

department: Biology

institute: Florida State University

first: Juan

middle: C

last: Rivera-Mulia


title: Effect of bacterial vaginosis, Lactobacillus and  Premarin estrogen replacement therapy on vaginal gene expression changes.

city: London

last: Carter

state: ON

address: 1151 Richmond Street North

department: London Regional Genomics Centre

institute: Robarts Research Institute

summary:
The aim of the study was to investigate gene expression profiles of post-menopausal women receiving Premarin estrogen replacement therapy (ERT), compared to controls and to examine any correlations between the bacterial vaginosis (BV) status of the stubjects.
This is the first study to use gene arrays to correlate changes in host expression to ERT and BV.

overall_design: Vaginal samples (8 control and 6 ERT) were collected at a single timepoint and processed for RNA recovery and Affymetrix array analysis, as well as Nugent scoring and denaturing gradient gel electrophoresis to identify bacteria.

first: Gregor

last: Reid

first: Kingsley

last: Anukam

first: Pirkka

last: Kirjavainen

first: Jo-Anne

last: Hammond

first: Sheri

last: Saunders

first: Adam

last: Dahn


title: RNA sequencing analysis of adult mixed phenotype acute leukemia (MPAL)

status: Public on Apr 25 2018

city: Houston

first: Koichi

last: Takahashi

state: TX

address: 1881 East Road

institute: UT MD Anderson Cancer Center

summary: RNA sequencing analysis was performed from bone marrow samples of 24 adult mixed phenotype acute leukemia (MPAL) patients

overall_design: RNA expression profiles of bone marrow samples from 24 adult mixed phenotype acute leukemia (MPAL) patients were generated by RNA-Seq using HiSeq 2000 platform.

first: Feng


title: Next Generation Sequencing Facilitates Quantitative Transcriptomes Analysis of  Smyd4 Downregulation HL-1 Cells By shRNA

first: deyong

last: xiao

address: Room 308, Mingdao building, Research Center For Birth Defects, School of Basic Medical Sciences,  Fudan University, 130 Dongan Road, Shanghai , China

institute: Fudan university

summary: The mRNA profiles of control cells and  cells of Smyd4 downregulation by shRNA were generated by deep sequencing.

overall_design: The mRNA profiles of control cells and  cells of Smyd4 downregulation were generated by deep sequencing using illumina

first: Deyong

last: Xiao

first: Duan

last: Ma


title: A Comparison of the Sexually Dimorphic Pravastatin Transcriptome in Mouse Cerebral Cortical Embryonic Neural Stem Cells

city: Pittsburgh

first: Uma

last: Chandran

state: PA

address: 5150 Center Ave

department: BioMedical Informatics

institute: University of Pittsburgh

summary: We used RNA-Seq analysis to identify the sexually dimorphic pravastatin regulated gene expression profile in primary cultures of mouse embryonic cerebral cortical neural progenitor/stem cells.

overall_design: RNAseq on primary E14.5 mouse cerebral cortical neurosphere cultures. 4 conditions - Male Cortex Pravastatin, Male Cortex Vehicle, Female Cortex Pravastatin, Female Cortex Vehicle. There are 3 biological replicates for each condition and all the 12 samples were run on two lanes (technical duplicates).

first: K

middle: A

last: Frahm

first: R

last: Carson

first: J

last: Waldman

first: A

last: Rudine

middle: P

last: Monaghan-Nichols

first: U

first: D

last: DeFranco


title: MicroRNAs mediate liver transcriptome changes upon soy diet intervention in mice

city: Timisoara

first: Edward

last: Seclaman

state: Timis

address: Burebista

country: Romania

institute: University Of Medicine and Pharmacy Timisoara

summary: Soy-based diets have triggered the interest of the research community due to their beneficial effects on a wide variety of pathologies like breast and prostate cancer, diabetes, and atherosclerosis. However, the molecular details underlying these effects are far from being completely understood and several recent attempts have been made to elucidate the soy-induced liver transcriptome changes in different animal models. Here we used Next Generation Sequencing to identify a set of two microRNAs specifically modulated by short-term soy-enriched diet in young male mice and estimate their impact on the liver transcriptome assessed by microarray. Clustering and topological community detection (CTCD) network analysis of STRING generated interactions of transcriptome data led to the identification of five topological communities of genes characteristically altered and putatively targeted by microRNAs upon soy diet intervention.

overall_design: Next Generation Sequencing was performed to measure the expression of microRNA species in the liver of mice fed for 4 weeks a soy-rich diet. Two groups of 3 mice each (12 weeks old mixed background males) were analysed.

first: Andrei

last: Anghel

first: Cristina

last: Bejinar

first: Ovidiu

last: Balacescu

first: Loredana

first: Mihai

last: Udrescu

first: Catalin

last: Marian

first: Ioan

middle: O

last: Sirbu


title: The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression [ELL2 rescue]

city: Lund

first: Björn

last: Nilsson

address: BMC

department: Hematology and Transfusion Medicine

institute: Lund University

summary: To understand the biological mechanism of ELL2 in multiple myeloma (MM), we show that the MM risk allele lowers ELL2 expression in CD138+ plasma cells (Pcombined=2.5×10-27; bcombined=-0.24 s.d.), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause-effect relationship.

overall_design: Reconstitution of ELL2 expression in L363-ELL2-knockout cells

first: Ram

last: Ajore


title: The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression

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

overall_design: Refer to individual Series


title: The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression [ELL2 KO]

overall_design: knock out ELL2 in L363 cells using CRISPR-Cas9

first: Mina

last: Ali


title: The role of CFTR in islet function

city: Nashville

first: Nathaniel

last: Hart

state: TN

address: 7465 Medical Research Building IV, 2215 Garland Ave.

department: Medicine

institute: Vanderbilt University Medical Center

summary: Cystic fibrosis (CF)-related diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting ~35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of b cell CFTR deletion, and normal and CF human pancreas and islets. Specific deletion of CFTR from murine b cells did not affect b cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein/electrical activity was not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion with few changes in key islet-regulatory transcripts. Furthermore, ~65% of b cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is not caused by intrinsic islet dysfunction from CFTR mutation, but rather, by b cell loss and intra-islet inflammation in the setting of a complex pleiotropic disease

overall_design: Human samples 3351-ACP-005, -0017, -0020, -0027, -0036 are isolated islets from normal healthy individuals. 3362-ACP-0011, -0012,-0013,-0014,-0015 are isolated from individuals with cystic fibrosis. 12 samples from mouse.

first: Alvin

last: Powers


title: Profiling of differential RNAs in LY500307-treated B16 cells and control-treated B16 cells

city: Chengdu

first: Shengtao

last: Zhou

address: #17, Renmin South Road

department: Department of Obstetrics and Gynecology

institute: West China Second Hospital, Sichuan University

summary: In this study, we examined the differential RNA profile of LY500307-treated B16 cells compared with control-treated B16 cells

overall_design: We used RNA sequencing to compare the differential RNAs of LY500307-treated B16 cells compared with control-treated B16 cells


title: Genome-wide transcriptome profiling of NEDD9-regulated genes using RNA-seq

city: Boston

first: Bradley

last: Maron

state: MA

address: 77 Avenue Louis Pasteur

institute: Brigham & Women's Hospital

summary:
Purpose:This study is to examine NEDD9-regulated and aldosterone-induced gene expression to understand the roles of NEDD9 and aldosterone in pulmonary arterial hypertension and pulmonary arterial fibrosis. The hypothesis tested here is whether there is a cross-talk between NEDD9 regulation and aldosterone signaling pathway in human pulmonary artery endothelial cells.
Methods: mRNA profiles of human pulmonary artery endothelial cells were generated by deep sequencing, in triplicate, using Ilumina Hiseq 2000. The sequence reads that passed quality filters were aligned to the human reference genome (hg19) using Tophat2.
Results: RNA-seq based transcriptome characterization revealed a number of NEDD9-regulated and aldosterone-induced profibrotic genes.
Conclusions: RNA-seq based transcriptome analysis helps to make biological discoveries and decipher complex biological functions.

overall_design: Transcriptome analysis was performed on human pulmonary artery endothelial cells. Six samples were transfected with NEDD9 siRNA and treated with aldosterone or vehicle control. 12 additional samples were directly treated with aldosterone or vehicle control.

first: Andriy

last: Samokhin


title: Human T-cell lymphoblastic lymphoma samples and control thymuses

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

country: Spain

institute: CNIO


title: RNA-Seq in human T-cell lymphoblastic lymphoma samples and control thymuses

summary: Precursor T-cell lymphoblastic neoplasms are aggressive haematological neoplasm that most often manifest with extensive marrow and blood affectation (T-cell acute lymphoblastic leukaemia or T-ALL) or less commonly as a thymic mass with limited bone marrow infiltration (T-cell lymphoblastic lymphoma or T-LBL). Here we show data from RNA-Seq in a sample series of T-LBL from Spanish patients.The goal was to determine the levels of expression of coding genes and microRNAs, and to identify all genetic variants including SNVs, indels, and fusion transcripts.

overall_design: Expression data were determined by comparson of each tumour sample with two control thymuses (404 and 405). Genetic variants were determined by comparison of tumour sequences with canonical ENSEMBL normal-references of each gene.

first: Jose

last: Fernandez-Piqueras

first: Pablo

last: Navarro

first: Pilar

last: Nieva

first: Maria

last: Morales

first: Marcos

last: Malumbres


title: Open chromatin landscape of mouse embryonic stem cells

status: Public on Apr 24 2018

city: La Jolla

first: Bing

last: Ren

state: CA

address: 9500 Gilman Dr., CMM-East, Admin Area/Rm 2071

institute: University of California, San Diego School of Medicine

summary: ATAC-seq data of E14 mouse embryonic stem cells

first: Hui

last: Huang


title: Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion

city: Amsterdam

first: Fleur

last: Peters

address: Meibergdreef 9

institute: Amsterdam UMC (AMC)

summary: Prior to (pre-) clinical application, MSC can be culture expanded as well as stimulated to activate their immunomodulatory properties. In the manuscript we show that stimulation of MSC induces immunophenotypical changes and marginally affects their DNA methylation pattern. Notably, culture expansion has a major impact on the DNA methylation profile of MSC, while their immunophenotype remained stable.

overall_design: Pure ucMSC samples of 4 donors were analyzed. ucMSC were stimulated with 3 treatments and 2 timepoints


title: NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence [RNA-Seq]

first: Shamith

last: Samarajiwa

address: ICTEM Building, Hammersmith Campus, Du Cane Road,

department: Genetics and Genomics Section  Department of Metabolism, Digestion and Reproduction

institute: Imperial College London

summary: We decribe the accessible chormatin landscape in RAS-induced (RIS) and NOTCH induced senescence (NIS) using ATAC-seq. By expressing active NOTCH (N1ICD) in the context of RIS, we find that N1ICD antagonises the formation of accessible regions in RIS. By performing co-cultures, we demonstrate that cells expressing a NOTCH1 ligand, JAGGED1, can antagonise the formation of RIS specific accessible regions.

overall_design: mRNA profiles were IMR90 cells expressing ER:HRAS(G12V) and a control vector or MSCV miR30 shHMGA1 were generated. 6 biological replicates.

first: Aled

last: Parry

first: Dóra

last: Bihary

first: Masashi

last: Narita


title: Parabiosis and single-cell RNA-Sequencing reveal a limited contribution of monocytes to myofibroblasts in kidney fibrosis

city: St. Louis

first: Haojia

last: Wu

address: 600 S Euclid Ave

department: Renal Division/Internal Medicine

institute: Washington University in St. Louis

summary: Fibrosis is the common final pathway of virtually all chronic injury to the kidney. While it is well accepted that myofibroblasts are the scar-producing cells in the kidney, their cellular origin is still hotly debated. The relative contribution of proximal tubular epithelium and circulating cells including mesenchymal stem cells, macrophages and fibrocytes to the myofibroblast pool remains highly controversial. Using inducible genetic fate tracing of proximal tubular epithelium we confirm that proximal tubule does not contribute to the myofibroblast pool. However, in parabiosis models in which one parabiont is genetically labeled and the other is unlabeled and undergoes kidney fibrosis, we demonstrate that a small fraction of genetically labeled renal myofibroblasts derive from the circulation. Single cell RNA-Sequencing confirms this finding but indicates that these cells are circulating monocytes, express few extracellular matrix or other myofibroblast genes and do express many proinflammatory cytokines. We conclude that this small circulating myofibroblast progenitor population contributes to renal fibrosis by paracrine rather than direct mechanisms.

overall_design: Single cell RNA-seq was performed on FACS-sorted PDGFRB+CD45- and PDGFRB+CD45+ cell populations

first: Benjamin

middle: D

last: Humphreys

first: Rafael

last: Kramann


title: Microarray: Discovery of a glucocorticoid receptor (GR) activity signature using selective GR antagonism in ER-negative breast cancer

city: Chicago

first: Suzanne

last: Conzen

address: KCBD 8th Floor Conzen Lab, 900 E 57th Street

institute: The University of Chicago

summary:
HGU133+2.0 Microarray on TNBC cells (MDA-MB-231) treated for 4, 8, or 12h with vehicle, 100nM dexamethasone, 100nM Dex/100nM Mifepristone, or 100nM Dex/100nM CORT108297
Purpose: Although high glucocorticoid receptor (GR) expression in early-stage estrogen receptor (ER)-negative breast cancer (BC) is associated with shortened relapse-free survival (RFS), how associated GR transcriptional activity contributes to aggressive BC behavior is not well understood. Using potent GR antagonists and primary tumor gene expression data, we sought to identify a tumor-relevant gene signature based on GR activity that would be more predictive than GR expression alone.  Design: Global gene expression and GR ChIP-sequencing were performed to identify GR-regulated genes inhibited by two chemically distinct GR antagonists, mifepristone and CORT108297. Differentially expressed genes from MDA-MB-231 cells were cross-evaluated with significantly expressed genes in GR-high versus GR-low ER-negative primary BCs. The resulting subset of GR targeted genes was analyzed in two independent ER-negative BC cohorts to derive and then validate the GR activity signature (GRsig).   Results: Gene expression pathway analysis of glucocorticoid-regulated genes (inhibited by GR antagonism) revealed cell survival and invasion functions. GR ChIP-seq analysis demonstrated that GR antagonists decreased GR chromatin association for a subset of genes. A GRsig comprised of n=74 GR activation-associated genes (also reversed by GR antagonists) was derived from an adjuvant chemotherapy-treated Discovery cohort and found to predicted probability of relapse in a separate Validation cohort (HR=1.9; p= 0.012).  Conclusions: The GRsig discovered herein identifies high-risk ER-negative/GR-positive BCs most likely to relapse despite administration of adjuvant chemotherapy. Because GR antagonism can reverse expression of these genes, we propose that addition of a GR antagonist to chemotherapy may improve outcome of these high-risk patients.

overall_design: MDA-MB-231 cells were treated with either Vehicle, 100 nM Dex +/- 100 nM C297 or 100 nM Mif for 4, 8, and 12h. Duplicate microarray experiments (n=2) for 4h treatments of Vehicle, Dex, and Dex/Mif conditions were performed along with a single experiment for the Dex/C297 treatment condition. 8h and 12h treatments for all samples were performed n=1 time.

first: Diana

last: West

first: Masha

last: Kocherginsky

first: Eva

last: Tonsing-Carter

first: Deniz

middle: N

last: Docen

first: David

last: Hosfield

first: Ricardo

last: Lastra

first: Jason

last: Sinnwell

first: Kevin

last: Thompson

first: Kathleen

last: Bowie

first: Ryan

last: Harkless

first: Maxwell

last: Skor

first: Charles

last: Pierce

first: Sarah

last: Styke

first: Caroline

last: Kim

first: Larischa

last: de Wet

first: Geoffrey

last: Greene

first: Judy

last: Boughey

first: Matthew

last: Goetz

first: Krishna

last: Kalari

first: Liewei

last: Wang

first: Gini

last: Fleming

first: Balázs

last: Győrffy


title: Gain of function of the ATXN1-CIC complex drives cerebellar pathology in Spinocerebellar ataxia type 1 (part 2)

city: Minneapolis

first: Christine

middle: M.

last: Henzler

address: 117 Pleasant St SE

department: Minnesota Supercomputing Institute

institute: University of Minnesota, Twin Cities (Minneapolis, MN, US)

summary: We isolated RNA from cerebella dissected from Pcp2-ATXN1[82Q], Pcp2-ATXN1[82Q]V591A;S602D, and wild-type littermates at one year of age

overall_design: Examination of gene expression in cerebellar lysates from two mouse groups

first: Maxime

last: Rousseaux

first: Ying-Wooi

last: Wan

first: Huda

middle: Y

last: Zoghbi

first: Harry

last: Orr


title: Gain of function of the ATXN1-CIC complex drives cerebellar pathology in Spinocerebellar ataxia type 1


title: Gain of function of the ATXN1-CIC complex drives cerebellar pathology in Spinocerebellar ataxia type 1 (part 1)

summary: We isolated RNA from cerebella dissected from En1-Cre; Cicflox/flox and  Cicflox/+ littermates at one year of age


title: Clinker: visualizing fusion genes detected in RNA-seq data

status: Public on Apr 23 2018

city: Parkville

first: Andrew

last: Lonsdale

state: VIC

address: 50 Flemington Rd

country: Australia

institute: Murdoch Children's Research Institute

summary: Genomic profiling efforts have revealed a rich diversity of oncogenic fusion genes, and many are emerging as important therapeutic targets. While there are many ways to identify fusion genes from RNA-seq data, visualising these transcripts and their supporting reads remains challenging. Clinker is a bioinformatics tool written in Python, R and Bpipe, that leverages the superTranscript method to visualise fusion genes. We demonstrate the use of Clinker to obtain interpretable visualizations of the RNA-seq data that lead to fusion calls. In addition, we use Clinker to explore multiple fusion transcripts with novel breakpoints within the P2RY8-CRLF2 fusion gene in B-cell Acute Lymphoblastic Leukaemia (B-ALL).

overall_design: RNA-seq of six B-cell Acute Lymphoblastic Leukaemia (B-ALL) samples with a P2RY8-CRLF2 fusion gene.

first: Paul

last: Ekert

first: Alicia

last: Oshlack


title: The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops [iCLIP]

status: Public on Apr 22 2018

city: Cleveland

first: Frank

last: Tedeschi

state: OH

address: 10900 Euclid AVE, cc/ Accounts Payable

institute: Case Western Reserve University

summary: The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and lncRNA function. How Dbp2p functions in these seemingly unrelated biological roles is not known. An important step towards addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using denaturing tandem affinity purification followed by UV-crosslinking. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and most markedly to non-coding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially interacts with sites near the 3’ ends. These sites closely correspond to regions where RNA-DNA hybrids (R-loop) form, and to binding sites of the RNA helicase Sen1p, a component of the Nrd1-Nab3-Sen1 (NNS) complex, which functions in transcription termination and 3' processing of non-coding RNAs in yeast. We also show that Dbp2p interacts in an RNA-independent manner with Sen1p in vivo. We further observe Dbp2p crosslinks to tRNAs and other RNAs also at sites where RNA-loops form. Collectively, our data link Dbp2p to diverse non-coding RNAs, to Sen1p, and to R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for seemingly unrelated cellular roles of Dbp2p.

overall_design: We investigated the cellular RNA binding sites of the DEAD-box helicase Dbp2p. This study contains two iCLIP and four RNA-seq datasets which includes a biological replicate.

first: Sara

last: Cloutier

first: Elizabeth

middle: J

last: Tran

first: Eckhard

last: Jankowsky


title: The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops


title: RNA sequencing of white adipose tissue of lean and obese mice

city: Oxford

first: Haifei

last: Shi

state: Ohio

address: 700 E High St

institute: Miami University

summary: We report differential expressed genes in white adipose tissue of mice fed with a low-fat diet or a high-fat diet.

overall_design: Methods: Male C57BL/6J mice at age of 6 weeks - 10 weeks fed either a low-fat diet or a high-fat diet.  mRNA profiles were generated by deep sequencing, in triplicates, using Illumina NextSeq500 sequencing system.  The sequence reads that passed quality filters were analyzed at the gene level with DESeq2.

first: Qi

last: Zhu

first: Jingyi

last: Cao

first: Lin

last: Liu

first: Benjamin

last: Hinkel

first: Bradley

last: Glazier

first: Chun

last: Liang


title: ERRγ coordinates a transcriptional program of mitochondrial and renal reabsorptive functions implicated in kidney disease [RNA-seq]

first: Grant

middle: D.

last: Barish

address: 303 E Superior Street

institute: Northwestern University

summary: Here we show that estrogen-related receptor γ (ERRγ) is an essential transcriptional coordinator of both renal mitochondrial and reabsorptive functions.  ERRγ is highly and specifically expressed in the RECs, and its expression correlates with kidney function in humans.  We generated REC-ERRγ KO mice which developed severe renal mitochondrial and reabsorptive dysfunction and fluid-filled cysts.  Transcriptome and cistrome analysis revealed that ERRγ directly regulates mitochondrial metabolism and renal reabsorption.  ChIP-reChIP-Seq studies further suggest that ERRγ employs a distinct mechanism to regulate renal reabsorption genes through functional cooperation with hepatic nuclear factor 1 beta (HNF1β), mutations of which cause strikingly similar renal dysfunction and cysts in humans and animals.  Together these findings reveal a novel role for ERRγ in simultaneously coordinating a transcriptional program for renal energy production via mitochondria and energy consumption to perform reabsorptive functions required for normal kidney function

overall_design: Identification of ERRg-regulated genes in mouse kidneys

first: Liming

last: Pei


title: RNAseq Study in CC-671 Treated Cal-51 Cells

first: Samuel

last: Danziger

address: 10300 Campus Point Dr

institute: Celgene

summary: CC-671 has been identified as an inhibitor of Cdc2-like kinase 2 (CLK2) and TTK in direct enzyme assays. CLK2 is a member of the CLK family that phosphorylates serine- and arginine-rich (SR) proteins of the spliceosomal complex as part of a regulatory mechanism for control of pre-mRNA splicing.  SR proteins are a family of small nuclear ribonucleoprotein particle (snRNP) splicing factors involved in constitutive and alternative splicing. Monitoring specific phospho-biomarkers of CLK2 demonstrated that CC-671 inhibited phosphorylation of CLK2 substrates in cancer cells with mean IC50 of 549 nM in the triple negative breast cancer (TNBC) line CAL51.  In this study, RNA sequencing approach was used to quantify the impact of CC-671 treatment on gene transcription and global alternative splicing in CAL51 cells.  Differential exon usage analysis demonstrated that CC-671 changed alternative splicing of many genes.  In addition, different sets of genes are impacted by CC-671 at both the alternative splicing and mRNA expression. Genes impacted by alternative splicing shared a set of common pathways with genes altered by mRNA expression. This result indicates that CC-671 regulates transcription via both gene expression and alternative splicing mechanisms.

overall_design: Triple negative breast cancer (TNBC) line CAL51 was grown in DMEM medium containing 10% fetal bovine serum, as recommended by vendor. The growing cells were treated by CC-671 in three biological replicates at the following concentrations and time intervals.  The treatment time points were 6 hour and 24 hour. Concentration of compounds used was 3 and 10 uM. Six million cells from each treatment were harvested and RNA was isolated by RNeasy kit. Poly-A selection and strand-specific RNA library construction were performed, followed by multiplexing indexed libraries and sequencing on the HiSeq 2500 with 2x100 bp read lengths. A total of 16 samples were included in this experiment, including 4 treatment groups with three biological replicates and 2 vehicle control groups with two biological replicates

first: Dan

first: Shuichan

last: Xu

first: Gordafaried

last: Deyanat-Yazdi

first: Sophie

middle: X

last: Peng

first: Leo

last: Barnes

first: Rama

last: Krishna Narla

first: Tam

first: David

last: Mikolon

first: Yuhong

last: Ning

first: Tao

first: Ning

last: Jiang

first: Heather

last: Raymon

first: Jennifer

last: Riggs

first: John

middle: F

last: Boylan


title: The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops [RNA-Seq]

first: Frank

last: Tedeschi

first: Sara

last: Cloutier

first: Elizabeth

last: Tran

first: Eckhard

last: Jankowsky