title: Post-translational buffering leads to convergent protein expression levels between primates

city: Chicago

first: Sidney

last: Wang

state: IL

address: 920 E. 58th Street, CLSC 317

department: Human Genetics

institute: University of Chicago

summary:
Differences in gene regulation between human and closely related species influence phenotypes that are distinctly human. While gene regulation is a multi-step process, the majority of research concerning divergence in gene regulation among primates has focused on transcription.
To gain a comprehensive view of gene regulation, we surveyed genome-wide ribosome occupancy, which reflects levels of protein translation, in lymphoblastoid cell lines derived from human, chimpanzee and rhesus macaque. We further integrated mRNA and protein level measurements collected from matching cell lines. We find that, in addition to transcriptional regulation, the major factor determining protein level divergence between human and closely related species is post-translational buffering. Inter-species divergence in transcription is generally propagated to the level of protein translation. In contrast, gene expression divergence is often attenuated post-translationally, potentially mediated through post-translational modifications.
Results from our analysis indicate that post-translational buffering is a conserved mechanism that led to relaxation of selective constraint on transcript levels in humans.

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens

overall_design: Using ribosome profiling assays, we measured levels of protein translation transcriptome-wide in lymphoblastoid cell lines derived from 4 HapMap Yoruba individuals, 5 chimpanzee individuals, and 5 rhesus macaque individuals. Note that the data uploaded for the 4 Yoruba individuals were generated by further sequencing the libraries generated as part of the Battle et al QTL mapping study (GSE61742). About 30~50% more data were generated for these individuals and the files uploaded here combined the new sequencing data with the old data from GSE61742 for each respective cell line.

middle: H

first: Yoav

last: Gilad

first: Jonathan

last: Pritchard

first: Joyce

last: Hsiao

first: Zia

last: Khan

first: Amy

last: Mitrano


title: RNA sequencing of plant microRNAs precipitated by the tombusviral suppressor of RNA silencing p19.

status: Public on May 09 2018

city: Halle

first: Claus

last: Weinholdt

address: Von-Seckendorff-Platz 1

department: Institute of Computer Science

country: Germany

institute: Martin Luther University Halle-Wittenberg

type: Non-coding RNA profiling by high throughput sequencing

summary:
Viral suppressors of RNA silencing, VSRs, counteract the antiviral RNA silencing of host plants by sequestration of virus-derived siRNAs. A central question concerns whether and how VSRs associate cellular miRNAs and thus modulate plant gene expression during a viral infection.
In this study we characterize the binding behaviour of the tombusviral p19 protein to miRNAs by performing an RNA-pull down experiment with bead-associated p19 protein from carnation italian ringspot virus. For this, we used cytoplasmatic extracs of Nicotiana tabacum protoplasts as an RNA source.
By applying Next Generation Sequencing (NGS) to the precipitated small RNAs, we were able to identify miRNAs specifically associating with the protein and other that were not efficiently bound by p19.

overall_design: Small RNAs were precipitated from Nicotiana tabacum cytoplasmatic BY2 lysate by carnation italian ringspot virus p19 protein. NGS-sequenced small RNAs were compared to the input RNA.

first: Robert

last: Pertermann

first: Selvaraj

last: Tamilarasan

first: Torsten

last: Gursinsky

first: Giorgio

last: Gambino

first: Jana

last: Schuck

first: Hauke

last: Lilie

first: Lena

last: Schwien

first: Ivo

last: Grosse

first: Ralph

last: Golbik

first: Vitantonio

last: Pantaleo

first: Sven-Erik

last: Behrens


title: Comparison of RNA expression of conditional Nmi knockout mammary epithelial cells to the wildtype control

city: Birmingham

first: Dongquan

last: Chen

state: AL

address: 1717 11th Ave South

department: Medicine

country: USA

institute: Univ of Alabama at Birmingham

type: Expression profiling by high throughput sequencing

summary: The goal of this study was to compare mRNA from mammary epithelial cells of 3 mammary-specific Nmi knockout FVB with corresponding wildtype control. This was performed to obtain clues to the signaling pathways that were impacted in the mammary epithelial cells upon knocking-out Nmi expression.

overall_design: To determine how the loss of Nmi contributed to a hyper-proliferative phenotype during puberty and lactation, we performed global RNAseq analysis from enriched mammary epithelial organoids from lactation day1 (L1), the time when Nmi protein expression in normal mammary epithelium is at its highest level. We compared 2 groups with 3 mice/group. We used second and third mammary glands of each mouse. These glands were isolated from mice on the first day of lactation, minced and dissociated in digestion medium (HBSS containing collagenase I (Sigma Aldrich, St. Louis, MO) (1mg/mL) and Pronase (Sigma Aldrich) (0.1mg/mL)) for two hours at 37C with shaking. Epithelial organoids were washed in PBS and enriched by pulse centrifugation to 1500rpm at least three times before subsequent assays.

first: Rajeev

middle: S

last: Samant


title: Protein kinase 7 regulating extracellular matrix assemblies and intracellular RAC1 and myosin II activities in the mesenchyme is important to Wolffian duct morphogenesis.

city: Charlottesville

first: Stephen

last: Turner

state: VA

address: 1670 Discovery Drive

institute: Signature Science, LLC

summary: Wolffian/epididymal duct morphogenesis highly coordinates with its specialized function of providing microenvironment for sperm maturation. Without normal development of Wolffian duct, male infertility will result. Therefore, it is important to understand the cellular and molecular mechanisms that regulated Wolffian duct morphogenesis. Our previous study showed that mediolateral intercalation of epithelial cells was a major driver of ductal elongation. In addition, radial intercalation of mesenchymal cells surrounding the duct also played roles in Wolffian duct morphogenesis, and all of these cell re-arrangements were regulated by protein tyrosine kinase 7 (PTK7), a member of the planar cell polarity (PCP) non-canonical Wnt pathway. In this study, we showed that PTK7 regulated assemblies of extracellular matrix (ECM) at the basement membrane and throughout the mesenchyme. Abnormal assembly of laminin, collagen IV, and nephronectin at the basement membrane and fibrosis-like deposition of fibrilla collagen in the interstitium were observed in Ptk7 knockout Wolffian ducts.  Meanwhile, PTK7 regulated activity levels of small GTPase RAC1 and myosin II in the mesenchyme of the Wolffian duct. Activity levels of RAC1 and myosin II decreased in the Ptk7 knockout mesenchyme compared to controls. When in-vitro-cultured Wolffian ducts were treated with collagenase IV, cross-link of fibrilla collagen was reduced, Wolffian duct elongation and coiling was reduced significantly, and cyst-like bulge was developed in the epithelial duct. When Wolffian ducts were treated with RAC1 inhibitor NSC23766, fibrilla collagen in the mesenchyme was disassembled, and Wolffian duct elongation was suppressed significantly. Our finding suggested that PTK7 regulated ECM assembly in the mesenchyme likely through regulating RAC1 and myosin II activities. Dynamic assembly and remodeling of ECM were important to Wolffian duct morphogenesis.

overall_design: Examination of gene expression in PTK7-/- Wolffian ducts

first: Bingfang

last: Xu

first: Barry

last: Hinton


title: In vivo CRISPR screening unveils multiple epigenetic regulators as novel tumour suppressor genes in lung cancer

city: Shanghai

first: Hongbin

last: Ji

address: 320 Yue Yang Road

country: China

institute: Shanghai Institute of Biochemistry and Cell Biology

summary: This study provides the first systematic CRIPSR screening of tumour suppressor genes (TSGs) in vivo and identifies bona fide TSGs especially for epigenetic regulators contributing to lung tumour progression. Moreover, our data provides a potential therapeutic strategy for the effective treatment of UTX-mutant lung tumours.

overall_design: mRNA profiles of Kras and Kras/Utx tumors were generated by next generation sequencing

first: Qibiao

last: Wu

first: Jian

last: Zhang

first: Hong

last: Zhao

first: Xinyuan

last: Tong

first: Ying

last: Tang

first: Lei

last: Gao

first: Zhaoyuan

last: Fang

first: Xin

last: Hu

first: Yahui

last: Tian

first: Fuming

last: Li

first: Zhen

last: Qin

first: Shun

last: Yao

first: Hsin-Yi

last: Huang

first: Kai

last: Ge

first: Luonan

first: Kwok-Kin

last: Wong

first: Liang


title: Small molecule inhibition of MEK activates Wnt signalling and leads to reprogramming of colon cancer stem cells [Illumina]

status: Public on May 08 2018

city: Heidelberg

first: Tianzuo

last: Zhan

address: Im Neuenheimer Feld 580

institute: German Cancer Research Center

summary: Resistance to Ras pathway inhibition is a major challenge in the treatment of colorectal cancer (CRC), but the underlying mechanisms are incompletely understood. Here we performed large-scale small molecule screens in CRC and identified inhibitors of MEK1/2 as potent activators of Wnt/beta-catenin signalling. Targeting MEK increased Wnt activity in different CRC cell lines and in the murine intestine in vivo. The MEK-induced Wnt response was strongly enhanced by truncating mutations in APC and proteomic experiments identified that AXIN1 levels are depleted upon MEK inhibition. We generated patient-derived colon cancer organoids and showed that a clinically approved MEK inhibitor leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stem cells and cancer relapse. This reprogramming was reverted by co-treatment with Wnt inhibitors. Our study demonstrates that MEK inhibition affects cellular plasticity and induces an intestinal stem cell program which constitutes a novel mechanism of drug resistance.

overall_design: Total RNA from human colorectal cancer organoids treated with trametinib for 72 h were compared to organoids treated with trametinib and PRI-724.

first: Benedikt

last: Rauscher

first: Michael

last: Boutros


title: Human serum and heparin-free platelet lysate as appropriate xeno-free alternatives for production of human MuStem cell batches

city: Nantes

first: Aurélie

last: Lardenois

address: INRA UMR 703 PAnTher, Ecole Nationale Vétérinaire, Agroalimentaire et de l'Alimentation, Nantes-Atlantique (Oniris) Atlanpole- La Chantrerie; CS. 40706

country: France

institute: INRA

summary:
Purpose: The population of muscle-derived stem cells called MuStem cells is presented as promising candidate for cell-based therapy of muscle diseases. To validate if this agent can be really presented as therapeutic product and so to be eligible to a future clinical use, it is now required to demonstrate beforehand an efficacy with cells prepared in compliance with good manufacturing practices (GMPs). The aim of the current study was to evaluate the use of two xeno-free blood derivatives corresponding to human serum (HS) and human platelet lysate (hPL) as alternatives to controverted but until now used fetal bovine serum (FBS) for isolation and expansion of human MuStem (hMuStem) cells.

Methods: A comparative study was performed with hMuStem cells isolated and in vitro expanded by using commercially available HS and hPL to determine its impact on their proliferation rates, clonogenicity, myogenic commitment level and oligopotency with regard to results obtained under FBS-based medium. Also, their respective phenotype and global gene expression patterns were investigated by flow cytometry and high throughput 3’ digital gene expression RNA-sequencing in order to define a possible differential impact of the human nutrients tested.

Results: Comparatively to FBS-based medium, use of HS- and hPL-supplemented ones efficiently supported long-term proliferation of hMuStem cells and enhanced clonogenicity, without main modification of their expression profile and allowing besides limiting the supplementation in growth factors. In vitro differentiation assay combined to transforming growth factor β1 (TGF-β1)-depletion experiments showed a lower myogenic commitment level as well as fusion ability of hMuStem cells when cultured with hPL-based medium according to a TGF-β1-independent process. Use of hPL-derived 3D hydrogel or fibrinogen-depleted hPL demonstrated that heparin-free hPL derivatives maintain consequent myogenic differentiation potential. In addition, the reduced myogenicity was shown to be rapidly reversible following replacement of hPL by HS or fibrinogen-depleted hPL.

Conclusions: All together, our original findings position HS and hPL as efficient and suitable alternatives to FBS for preparation of hMuStem cell batch in compliance with GMPs.

overall_design: mRNA profile of hMuStem cells cultured in hPL was compared to the mRNA profile of hMuStem cells cultured in HS. The profiles were generated in triplicates using the 3'DGE-Seq technology.

first: Aurelie

first: Charlotte

last: Saury

first: Karl

last: Rouger


title: Transciptomic analysis of maize response to the attack of Sesamia nonagrioides

city: Pontevedra

first: Victor

middle: M

last: Rodriguez

address: Apartado 28

department: Plant Genetics and Breeding

country: Spain

institute: Mision Biologica de Galicia (CSIC)

summary: We performed a transcriptomic analysis to identify genes differentially transcribed in the maize stem upon corn borer feeding and treatment with insects regurgitates by using the MACE (Massive Analysis of cDNA Ends) technology.

overall_design: Two comparisons were performed: Insect chewing vs control and Regurgitate+wounding vs wounding in three biological replicates per treatment

first: Guillermo

last: Padilla

first: Rosa

middle: A

last: Malvar

first: Mario

last: Kallenbach

first: Rogelio

last: Santiago

first: Ana

last: Butron


title: Small molecule inhibition of MEK activates Wnt signalling and leads to reprogramming of colon cancer stem cells

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

overall_design: Refer to individual Series


title: Atrial Molecular Asymmetry Precedes the Emergence of Cardiac Septation [RNA-seq]

city: Bad Nauheim

first: Almary

middle: Victoria

last: Guerra Rodríguez

state: Hessen

address: Ludwigstrasse 43

department: Dept. III Developmental Genetics

institute: Max Planck Institute for Heart and Lung Research

summary: Comparison of the meis2b+ and the meis2b- halves of the atrium of the adult zebrafish atrium reveals the existence of two different transcriptional domains.  These two domains analogous to that of the two atria in terrestrial vertebrates

overall_design: To determine the expression profiles of the Tg(meis2b-reporter)-positive vs -negative atrial compartments, a total of 6 hearts of 3 mpf Tg(meis2b-reporter) zebrafish were micro-dissected.  A total of 4 pools were made: the first two pools, each contained 3 Tg(meis2b-reporter)-positive atrial compartments, and the other two contained the Tg(meis2b-reporter)-negative halves.

last: Guerra

first: Raoul

middle: F

last: Germano

first: Oliver

last: Stone

first: Rima

last: Arnaout

first: Benoit

last: Vanhollebeke

first: Stefan

last: Guenther

first: Suchit

last: Ahuja

first: Didier

middle: Y

last: Stainier

first: Sven

last: Reischauer


title: Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP

city: St. Lucia

first: Othmar

last: Korn

state: Queensland

address: University of Queensland

country: Australia

institute: Australian Institute for Bioengineering and Nanotechnology

summary: Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4 or 5448-infected TEpi cells, were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2 and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.

overall_design: 12 samples, from primary human tonsil surface and crypt epithelial cells

first: Amelia

middle: T

last: Soderholm

first: Timothy

middle: C

last: Barnett

first: Tania

last: Rivera-Hernandez

first: Lisa

last: Seymour

first: Benjamin

middle: L

last: Schulz

last: Nizet

first: Christine

last: Wells

first: Matthew

middle: J

last: Sweet

first: Mark

last: Walker


title: Small molecule inhibition of MEK activates Wnt signalling and leads to reprogramming of colon cancer stem cells [Affymetrix]

first: Tianzuo

last: Zhan

overall_design: Total RNA from human colorectal cancer organoids treated with trametinib for 72 h.

first: Benedikt

last: Rauscher

first: Michael

last: Boutros


title: RNA-seq to compare wild type, db/+ and db/db islets

city: Nedlands

first: Abraham

middle: Neelankal

last: John

state: WA

address: 6, verdun st

department: Islets cell development

institute: Harry Perkins Institute of Medical Research

summary:
Purpose: RNA seq analysis were to compare and contrast the gene expression profile involved in the dedifferentiation of db/db islets in type 2 diabetes
Methods: Islets of wild type, db/+ and db/db were purified using perfusion from 12 week old mice and RNA were isolated. Islated RNA were used in RNA seq to understand the expression pattern
Results: Using an optimized data analysis workflow, we mapped about 10 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts WT, db/+ and db/db mice islets with TopHat workflow. Hierarchical clustering of differentially expressed genes uncovered there role in type 2 diabetes. Data analysis with TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.
Conclusions: We characterised and identified genes involved in dedifferentiation of islets.

overall_design: Islets of mRNA 12 weeks old wild type (WT), db/+ and db/db mice were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 platform.

first: Ramesh

last: Ram

first: Fang-Xu

last: Jiang


title: Gene Regulation by Histone Deacetylase 7 During Invariant Natural Killer T Cell Development

city: Novato

first: Eric

last: Verdin

state: CA

address: 8001 Redwood Blvd

institute: Buck Institute for Research on Aging

summary: We have found that the signal-dependent chromatin modulator Histone Deacetylase 7 interacts with and corepresses the signature transcription factor of invariant natural Killer T (iNKT) cells, ZBTB16. This study was designed to determine the global effect on transcription of a gain-of-function mutant of HDAC7 during iNKT cell development and function.  Va14/Ja18 T cell receptor transgenic thymocytes and splenocytes, which develop preferentially into iNKT cells, were profiled by RNA-seq with and without expression of a gain-of-functon HDAC7 transgene (HDAC7dP)

overall_design: There are 18 samples overall, consisting of 3 replicates each of CD4 splenoctes and CD4SP thymocytes, with either no transgene or with the V-alpha14/j-alpha18 TCR transgene +/- the HDAC7 deltaP transgene.  The Va14/Ja18 TCR transgenic cells were sorted using CD1D/aGalCer tetramers to identify NKT cells.

first: Herbert

middle: G

last: Kasler

first: Intelly

last: Lee

first: Hyung

middle: W

last: Lim


title: RNA-Seq experiments performed on rat Schwann (S16) cells

city: Ann Arbor

first: Anthony

last: Antonellis

state: MI

address: 1241 E Catherine St.

institute: University of Michigan

summary: We utilized RNA-Seq on rat Schwann (S16) cells to determine global gene expression. This information was generated as part of a larger effort to characterize cis-regulatory elements and global gene expression within Schwann cells. To achieve this, we generated RPKM values across two independent biological replicates. This dataset was also used to predict cis-regulatory element function on genes following CRISPR knockout studies.

overall_design: Performed two technical replicates of RNA-Seq on two independent biological replicates of S16 cells

first: William

middle: D

last: Law


title: Transcriptomics of Dorso-Ventral axis determination in Xenopus tropicalis

status: Public on May 04 2018

city: London

first: Rita

last: Monteiro

address: 1 Midland Road

department: Developmental Biology Laboratory

country: United Kingdom

institute: The Francis Crick Institute

summary: We studied the transcriptomic differences between ventralised and dorsalised Xenopus tropicalisembryos as a result of UV irradiation and LiCl treatment, respectvely. Both manipulations have been used by researchers to perturb the establishment of the dorso-ventral axis, however the whole transcriptomes of the resulting embryos have never been fully characterised or compared. We show that ventralized embryos are transcriptionally closer related to untreated embryos than dorsalized embryos. Furthermore comparison to dissected ventral and dorsal parts of an unperturbed gastrula embryo indicates that UV and LiCl treatment indeed enriches for ventral and dorsal cells, respectively.

overall_design: Comparison of poly(A) transcriptome between Xenopus tropicalis LiCl-, UV-treated and untreated gastrulae embryos. For each condition there are five biological replicates that were used for the analyses.

first: James

last: Smith

first: George

middle: E

last: Gentsch


title: mRNA-seq Whole Transcriptome Profiling of Fresh Frozen versus Archived Fixed Tissues

status: Public on May 03 2018

city: Rehovot

first: Noa

last: Bossel Ben-Moshe

address: Hertzl Street

country: Israel

institute: Weizmann

summary:
Background: The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging.
Results: In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions.
Conclusions: Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

overall_design: We perform an unbiased evaluation of RNA-seq of archived tumor tissues by comparing the same library preparation methods for both FF and FFPE matched tumor samples and for small amounts of total RNA starting material. We have 3 matched FF/FFPE tumor samples with a moderate archival time of about 4-5 years (T1=T3), and additional 3 FFPE tumor samples archived for more than 10 years (T4-T6). all samples were tested with two protocols: illumina Truseq RNA after poly(A) selection (mRNA-seq); and Truseq after ribosomal depletion (RiboZero). Several initial amounts of starting material was tested for eacg protocol.

first: Shlomit

last: Gilad

first: Gili

last: Perry

first: Sima

last: Benjamin

first: Nora

last: Balint-Lahat

first: Anya

last: Pavlovski

first: Sharon

last: Halperin

first: Barak

last: Markus

first: Ady

last: Yosepovich

first: Iris

last: Barshack

first: Einav

middle: N

last: Gal-Yam

first: Eytan

last: Domany

first: Bella

last: Kaufman

first: Maya

last: Dadiani


title: Dissecting the functions of mycobacteria RbpA structural domains

city: Saint Louis

first: Christina

last: Stallings

state: Missouri

address: 660 S. Euclid Avenue

institute: Washington University in St Louis

summary: RNA polymerase (RNAP) binding protein RbpA contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Based on structural and biochemical data, four domains have been identified in the RbpA protein: a N-terminal tail (NTT) domain of unknown function, a core domain (CD) that contacts the RNAP β’ subunit in a recently solved crystal structure, a basic linker (BL) that binds DNA, and a s-interaction domain (SID) that binds group I and group II s-factors. However, limited in vivo studies have been performed in mycobacteria and how the individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We dissected the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis. We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP holoenzyme, indicating that the primary function of the CD is not solely association with the RNAP. We show that RbpA BL and SID are required for stabilization of RPo complexes at the ribosomal RNA rrnAP3 promoter in vitro, while the NTT and CD are dispensable. Finally, we determine that the NTT and CD impact gene expression of a distinct set of genes from that affected by the BL and SID activities. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.

overall_design: Transcriptomes of 4 Mycobacterium smegamtis strains including csm275 (WT RbpA), csm314 (RbpA R88A), csm322 (RbpA R79A) and csm328 (RbpA delta 1-71), which express different RbpA alleles are compared in triplicate.

first: Jerome

last: Prusa


title: Simultaneous and systematic analysis of cellular and viral gene expression during Enterovirus 71-induced host shutoff

city: Tianjin

first: Yongquan

last: Lin

address: No. 94, Weijin Road

institute: Nankai University

summary: Enterovirus 71 (EV71) infection causes a profound shutoff of cellular protein synthesis. Deep RNA-sequencing and ribosome profiling were employed to systematically analyze messenger RNA and ribosome-protected RNA in EV71-infected rhabdomyosarcoma cells at progressive time points following infection. The analysis characterized the dynamic transcriptional and translational landscapes of both the virus and host cells. The results indicated that reduced translation of cellular mRNAs played a key role in EV71-induced host shutoff rather than mRNA depletion. During the host shutoff, EV71 protein was preferably synthesized through both a translational advantage and abundant mRNA production. Moreover, a small number of cellular genes were resistant to the host shutoff through both transcriptional and translational regulation, including genes in mitogen-activated protein kinase (MAPK) signaling pathway that is important for EV71 replication. These results indicated selective cellular protein synthesis during EV71-induced host shutoff as a mechanism the virus utilizes to benefit its replication.

overall_design: Ribosome profiling and RNA-Seq of RD cell infected with EV71 or mock, 3 time points, two triplicate in the last two time points, using Illumina Hiseq 2000.

first: Yan

last: Wang

first: Zhi

last: Xie

first: Juan

last: Tan

first: Zhilong

last: Yang


title: The oligodendrocyte "protrusiosome": Identification of compartmentalized enrichment of transcripts in newly-formed protrusions of oligodendrocyte precursor cells (OPC) by RNAseq

status: Public on May 07 2018

city: Porto

first: Helena

middle: Sofia

last: Domingues

address: Rua Alfredo Allen, 208

department: Neurobiology and Neurologic Disorders

country: Portugal

institute: Instituto de Investigação e Inovação em Saúde

summary: Like neurons, oligodendrocytes (OL) are cells with elaborate morphology that probably require asymmetrical spatial regulation of biological processes.  Formation of membrane protrusions is critical for OL development and interaction with axons. We hypothesized that the enrichment of specific mRNAs in protrusions of oligodendrocyte precursor cells (OPC) is important for morphological differentiation, thus having an impact in myelination.  To explore this hypothesis, we established a modified Boyden chamber system to physically separate soma from membrane protrusions of rat primary OPC cultured in vitro for 24h. We performed a whole transcriptome analysis (RNAseq) of primary rat OPC soma and membrane protrusion fractions and found a subcellular enrichment of mRNAs in these structures during initial protrusion formation. At the very initial stage of OPC protrusion extension, there is a significant subcellular enrichment of transcripts encoding proteins related to cellular component assembly and cytoskeleton organization, particularly of actin-related molecules.  This suggests that the regulation of the cytoskeleton dynamics may be locally controlled in OPCs and probably relevant for their differentiation program.

overall_design: Rat OPCs were plated on a modified Boyden chamber system for 24h consisting of a transwell with 1μm pore size with chemo- and  haptotactic gradients to physically separate OPC soma and protrusion fractions for RNA sequencing. RNAseq was done on 2 samples for OPC soma and 2 samples of OPC protrusions.  Due to very low RNA yields in OPC protrusions samples (typically 9ng per well), each sample consisted in a pool of several experiments.

first: João

middle: B

last: Relvas


title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination

city: Oxford

first: Nicholas

last: Proudfoot

address: South Parks Road

institute: Sir William Dunn School of Pathology,  University of Oxford

summary: We performed high-throughput sequencing of chromatin-associated RNA from CH12 cells depleted of Ddx1 and induced to undergo Class Switch Recombination (CSR).

overall_design: CH12 cells depleted for Ddx1 (shDdx1) and control (shCtrl), cultured in unstimulated (UNS) and CSR inducing (CIT) conditions; 2 replicates each sample.

first: Claudia

last: Ribeiro de Almeida

first: Somdutta

last: Dhir

first: Ashish

first: Amin

last: Moghaddam

first: Quentin

last: Sattentau

first: Anton

last: Meinhart


title: RNA-seq-based Transcriptome Analysis of A LuxR-like (DqsR) Mutant of Deinococcus radiodurans Under Oxidative and Non-oxidative Stresses.

city: Hangzhou

first: Lin

state: Zhejiang

address: No.268, Kaixuan Road

department: Institute of Nuclear-Agricultural Sciences

institute: Zhejiang University

summary:
Purpose: The goal of this study is to explore the role of DqsR of D. radiodurans R1 in transcriptome profiling under oxidative and non-oxidative stresses.
Methods: mRNA profiles of wild-type D. radiodurans R1 and DqsR knockout strain with 100 mM H2O2 treatment or not were generated by deep sequencing using IIllumina Solexa.
Results: A total of 17.03 million (M) and 19.61 M clean reads were obtained from the wild-type R1 and mutant strains; of these, 14.81 M and 16.78 M reads were mapped to the genome, respectively. A total of 427 genes were repressed and 396 genes were induced at least one-fold (log2) (p<0.05) in the mutant under non-oxidative stress conditions in transcription.
Conclusions: Our study reveals that deletion of DqsR changes the transcriptional levels of a number of genes, both under normal conditions and oxidative stresses, including genes involved in translation, transcription, replication, recombination and repair, inorganic ion transport and metabolism, etc.

sample_organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539

overall_design: Examination of mRNA levels in wild-type D. radiodurans R1 and the DqsR mutant under oxidative and non-oxidative stresses by deep sequencing using Illumina Solexa.

first: Bing

last: Tian


title: Age-related Islet Inflammation Marks the Proliferative Decline of Pancreatic Beta-cells in Zebrafish

city: Dresden

first: Nikolay

last: Ninov

address: Fetscherstrasse 105

institute: CRTD

summary: Individual organisms age at different rates, however, it remains unclear how aging alters the properties of individual cells.  Here we show that zebrafish pancreatic beta-cells exhibit heterogeneity in both gene expression and proliferation with age.  Individual beta-cells show marked variability in transcripts involved in endoplasmic reticulum stress, inhibition of growth factor signaling and inflammation, including NF-kB signaling.  Using a reporter line, we show that NF-kB signaling is indeed activated heterogeneously with age.  Notably, beta-cells with higher NF-kB activity proliferate less compared to neighbors with lower activity.  Furthermore, NF-kB-signalinghigh beta-cells from younger islets upregulate socs2, a gene naturally expressed in beta-cells from older islets.  In turn, socs2 can inhibit proliferation cell-autonomously.  NF-kB activation correlates with the recruitment of tnfα-expressing immune cells, pointing towards a role for the islet microenvironment in this activity.  We propose that aging is heterogeneous across individual beta-cells and identify NF-kB signaling as a marker of heterogeneity.

overall_design: We used fluorescence-activated cell sorting (FACS) coupled with next generation RNA-Sequencing to profile beta-cells from 3 month post fertilization and 1 year post fertilization animals. total RNA was extracted from FACS sorted beta-cells using Quick-RNA MicroPrep kit (R1050 Zymo Research). Sequencing was performed on llumina HiSeq2500 in 2x75bp paired-end mode. Reads were splice-aligned to the zebrafish genome, GRCz10, using HISAT2. htseq-count was used to assign reads to exons thus eventually getting counts per gene.

first: Sharan

last: Janjuha

first: Sumeet

middle: P

last: Singh


title: Alterations in retrotransposon control lead to a wide gene reprogramming in tobacco

city: Sao Paulo

first: Erika

middle: Maria

last: de Jesus

state: SP

address: Rua do Matao, 277, sala 153

department: Botany

country: Brazil

institute: University of Sao Paulo

summary: We sequenced mRNA from leaves of 4 N. tabacum lineages, one control and biological replicates of mutants presenting a chlorotic spots phenotype on leaves resultant from the interference in Tnt1 retrotransposon expression, in order to identify genes co-regulated with Tnt1

overall_design: Examination of mRNA levels in control and mutant tobacco leaves

first: E

middle: M

first: M

middle: A

last: Van Sluys


title: Single‐cell transcriptomic analysis of mouse CA1 inhibitory neurons

status: Public on May 11 2018

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: We  studied  the  transcriptomes of mouse CA1 inhibitory cells. Novel clustering methods identified all 23 previously  described  CA1  inhibitory  types,  while  also  suggesting  6  new  inhibitory  classes.  Latent‐factor  analysis revealed a common continuum of expression of many genes within and between classes,  which  we  hypothesized  correlates  with  a  continuum  from  faster‐spiking  cells  that  proximally  target pyramidal cells, to slower active cells targeting pyramidal distal dendrites or interneurons.

overall_design: 6971 single cells isolated from the mouse cortex CA1 region

first: Kenneth

last: Harris

first: Carolina

last: Bengtsson Gonzales

first: Hannah

last: Hochgerner

first: Nathan

last: Skene

first: Lorenzo

last: Magno

first: Linda

last: Katona

first: Peter

last: Somogyi

first: Nicoletta

last: Kessaris

first: Jens

last: Hjerling-Leffler


title: Cooptation of tandem DNA repeats for the control of epithelial-to-mesenchymal transition [RNA-Seq]

status: Public on May 02 2018

city: Milan

first: Chiara

last: Balestrieri

address: Via Olgettina 58

department: Center for Omics Sciences

country: Italy

institute: IRCCS San Raffaele Scientific Institute

summary: During normal or pathological epithelial-to-mesenchymal transition, epithelium-specific gene expression is shut down, with the DNA-binding factor ZEB1 acting as a master suppressor of epithelial identity. Here, we show that ZEB1 occupies primate-specific tandem repeats (TRs) harboring dozens of copies of its DNA-binding motif and located within genomic loci relevant for epithelial identity. Deletion of one such repeat in a quasi-mesenchymal human cancer cell line induced the reacquisition of epithelial features and phenocopied the effects of ZEB1 gene deletion. Since ZEB1 binds clustered motifs in a non-cooperative manner, changes in its nuclear concentration enable graded adjustments of TR occupancy, thus fine-tuning repression level. In addition, high motif density in TRs allows ZEB1 binding (and shutdown of epithelial programs) despite differences in chromatin organization and accessibility among epithelial cell types.

overall_design: Total RNA from human pancreatic ductal adenocarcinoma cell lines was processed for multiparallel sequencing. Experiments were carried out in genome edited clonal MiaPaCa2 cells (3 ZEB1-deleted CRISPR-Cas9 clones and 3 wt clones).

first: Gioacchino

last: Natoli


title: RTP determination in cell lines

status: Public on May 01 2018

first: Cheng

last: Zhang

address: Tomtebodavägen 23A, Solna

department: Division of Proteomics and Nanobiotechnology

institute: Royal Institute of Technology, KTH

summary: Nine cell lines were analyzed for transcriptomics and proteomics analyses. We wanted to measure the ratio between protein and mRNA in different cell lines and see if it is conserved across them.

overall_design: We performed parallel transcriptomics and proteomics analyses in three different biological replicates in nine cell lines derived from different organs.  This series contains only the transcriptome data.

first: Gianluca

last: Maddalo

first: Fredrik

last: Edfors


title: Characterization of a spontaneous model of inflammation-derived liver cancer [ChIP-seq]

city: Rozzano

first: Alberto

last: Termanini

address: Via Manzoni, 113

institute: Istituto Clinico Humanitas

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Induction of a protective response to oxidative stress, both from a transcriptional and from a genetic viewpoint, confers a selective advantage for cells exposed to an inflammatory environment, paving the way to neoplastic transformation

overall_design: Chromatin immuno-precipitations of acetylated lysine 27 of H3 followed by multiparallel sequencing, performed on tissues of Mdr2-WT (FVB.129P2) livers, Mdr2-KO (FVB.129P2-Abcb4tm1Bor/J) inflamed livers or hepatocarcinoma nodules from Mdr2-KO livers. All samples have been treated with clodronate liposomes to prevent sample contamination by liver macrophages

first: Agnese

last: Collino

first: Serena

last: Ghisletti


title: Next Generation Sequencing of  skin from wild-type and ADAM17 cKO (Sox9-Cre x ADAM17fl/fl) mice

city: Bethesda

first: Tetsuro

last: Kobayashi

state: MD

address: 9000 Rockville Pike, Bldg 10, Rm12N256

institute: NIAMS/NIH

summary:
Purpose: Transcriptomic analysis of ADAM17 cKO mice, a mouse model of atopic dermatitis.
Methods: Skin was harvested from 10-week-old wild-type and ADAM17 cKO mice and were subjected to next generation sequencing as described.
Results: Of 13,573 genes analyzed by GSA, 2514 differentially expressed genes (DEGs) were detected in ADAM17 cKO skin using a standard definition including both statistically significant change (P-value < 0.01; FDR < 0.05) and effect size (|fold change| > 2) . These DEGs included genes important in lipid metabolism and keratinization, consistent with the understanding of barrier dysfunction in atopic inflammation, as well as significant immunologic pathways in adaptive and innate immunity previously identified in the human AD transcriptome
Conclusions: Our study reveals that the transcriptome of ADAM17 cKO mice capture a part of upregulated genes previously observed in human AD skin.

overall_design: Epidermal and whole skin mRNA profiles of 10-week-old WT and ADAM17 cKO mice were generated by deep sequencing, in triplicates.

first: Keisuke

last: Nagao


title: Characterization of a spontaneous model of inflammation-derived liver cancer

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

overall_design: Refer to individual Series


title: Next Generation Sequencing Analysis of B16F10.9 melanoma cells untreated or treated with IFN-gamma

city: New york

first: Ariella

last: Glasner

state: NY

address: 504 E 89th Street, Apt 3A

department: Immunology

institute: MSKCC

summary: We compare gene expression in B16F10.9 melanoma cells untreated or treated with IFN-gamma

overall_design: mRNA profiles of B16F10.9 melanoma cells untreated or treated with IFN-gamma were generated by deep sequencing, in triplicates.

first: Ofer

last: Mandelboim


title: De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia

city: Lund

first: Axel

last: Hyrenius Wittsten

address: Klinikgatan 28

department: Division of Clinical Genetics

institute: Lund University

summary: Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3ITD, FLT3N676K, and NRAS G12D accelerate KMT2A-MLLT3 leukemia onset.  Subclonal FLT3N676K mutations also accelerate disease, possibly by providing stimulatory factors such as Mif. Acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 were identified in KMT2A-MLLT3 leukemia cells and favored clonal expansion. During clonal evolution, serial genetic changes at the KrasG12D locus was observed, consistent with a strong selective advantage of additional KrasG12D. KMT2A-MLLT3 leukemias with signaling mutations enforced Myc- and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlights the importance of activated signaling as a contributing driver in this disease.

overall_design: mRNA sequencing of KMT2A-MLLT3 leukemias with or without activating mutations generated using Illumina NextSeq 500.

last: Hyrenius-Wittsten

first: Anna

middle: K

last: Hagström-Andersson


title: CBFb-SMMHC inhibition triggers apoptosis by disrupting MYC chromatin dynamics in acute myeloid leukemia [RNA-seq]

city: Worcester

first: Lucio

last: Castilla

state: MA

address: 364 Plantation Street

department: MCCB

institute: University of Massachusetts

summary: We recently reported the discovery of a small molecule inhibitor, AI-10-49 which can specially inhibit the protein-protein interaction between RUNX1 tumor suppressor and CBFβ-SMMHC oncogene. We also demonstrated that AI-10-49 can re-establish the RUNX1 transcriptional program in inv(16) cells and can extend the survival of inv(16) leukemic mice. To identify the transcriptional changes associated with AI-10-49, we performed RNA-seq analysis in ME-1 cells [human inv(16) leukemia cell line] treated with AI-10-49.

overall_design: ME-1 cells were treated with DMSO/ AI-10-49 (1uM) for six hours, followed by RNA isolation. RNA libraries were sequenced using 90bp paired end reads on an Illumina HiSeqTM 2000.Three independent experiments were conducted for DMSO as well as AI-10-49 treatments.

first: John

last: Pulikkan

first: Mahesh

last: Hegde

first: A

last: Ahmad

first: Houda

last: Belaghzal

first: Anuradha

last: Illendula

first: Jun

last: Yu

first: Kelsey

last: O’Hagan

first: J

last: Ou

first: C

last: Muller-Tidow

first: Scot

last: Wolfe

first: Lihua

last: Zhu

first: Job

last: Dekker

middle: H

last: Bushweller


title: A de novo mouse model of C11orf95-RELA fusion-driven ependymoma identifies driver functions in addition to NFκB

city: Seattle

first: Sonali

last: Arora

address: 1100 Fairview Ave N,

institute: FHCRC

summary: The vast majority of supratentorial ependymomas (ST-EPNs) have few mutations other than chromosomal rearrangements on chromosome 11, most generating a fusion between C11orf95 and RELA (CR). This CR fusion can transform stem cells ex vivo rendering them oncogenic and may possess NF-κB activity, which has been proposed to be a mechanism of oncogenesis. However, it is not known whether CR is sufficient for EPN formation in vivo, and from what cell type and location. We found that CR is sufficient to form tumors from cells in the ependymal zone in mice that show many molecular and histologic similarities to human ST-EPN. Furthermore, the activation of NF-κB by this fusion protein appears minimal and not related to its oncogenic activity

overall_design: C11orf95-RELA is a potent oncogene for supratentorial ependymoma

first: Tatsuya

last: Ozawa

first: Frank

last: Szulzewsky

first: Gordana

last: Juric-Sekhar

first: Yoshiteru

last: Miyajima

first: Hamid

last: Bolouri

first: Yoshie

last: Yasui

first: Jason

last: Barber

first: James

last: Dalton

first: Terreia

middle: S

last: Jones

first: Mitsutoshi

last: Nakada

first: Toshihiro

last: Kumabe

first: David

last: Ellison

first: Richard

last: Gilbertson

first: Eric

middle: C

last: Holland


title: Characterization of a spontaneous model of inflammation-derived liver cancer [RNA-seq]

first: Alberto

last: Termanini

overall_design: Sequencing of RNA extracted from tissues of Mdr2-WT (FVB.129P2) livers, Mdr2-KO (FVB.129P2-Abcb4tm1Bor/J) inflamed livers or hepatocarcinoma nodules from Mdr2-KO livers. All samples have been treated with clodronate liposomes to prevent sample contamination by liver macrophages

first: Agnese

last: Collino

first: Serena

last: Ghisletti

first: Gioacchino

last: Natoli


title: Toll-Like Receptor 7 Agonist GS-9620 Induces Prolonged Inhibition of HBV in Human Hepatocytes via a Type I Interferon-Dependent Mechanism

city: Foster City

first: Li

last: Li

address: 333 Lakeside Drive

institute: Gilead Sciences

summary: TLR7 induced transcriptional change in HBV-infected primary human hepatocytes

overall_design: RNA-Seq was conducted in two independent primary human hepatocyte (PHH) donors. After day 13 post-infection, primary human hepatocytes (PHH) were infected with HBV and treated with GS-9620, with media from human peripheral blood mononuclear cells (PBMCs) treated with GS-9620 (GS-9620 conditioned media; GS-9620-CM)

first: Congrong

last: Niu

first: Stephane

last: Daffis

first: Julie

last: Lucifora

first: Marc

last: Bonnin

first: Sarah

last: Maadadi

first: Eduardo

last: Salas

first: Hilario

last: Ramos

last: Durantel

first: Fabien

last: Zoulim

first: William

last: Delaney IV

first: Simon

last: Fletcher


title: Maternal Folate Genes and Aberrant DNA Hypermethylation among Offspring with Pediatric Lymphoblastic Leukemia

city: Houston

first: Jeremy

last: Schraw

state: TX

address: 1 Baylor Plaza MS:305

institute: Baylor College of Medicine

type: Methylation profiling by high throughput sequencing

summary: Pediatric acute lymphoblastic leukemia (ALL) is believed to originate in utero and frequently involves aberrant promoter methylation. Folate is the methyl donor for DNA methylation, suggesting that maternal folate metabolism may contribute to the development of ALL. We previously reported significant associations between single nucleotide polymorphisms (SNPs) in the maternal methionine synthase (MTR) gene and offspring’s risk of ALL. Here, we test the associations of 11 SNPs in MTR with aberrant DNA methylation in offspring with ALL. We recruited 51 ALL case-mother pairs from Texas Children’s Hospital from 2005-2010. We collected maternal saliva samples and diagnostic bone marrow plasma from cases. Bone marrow plasma was obtained from six healthy donors. DNA methylation was determined using MCA-Seq. Pyrosequencing was used to determine maternal MTR genotypes. We identified offspring with high and low promoter methylation and used logistic regression to estimate the effects of maternal genotype on offspring methylation. Twenty-two cases (43%) demonstrated high promoter methylation. Maternal MTR 113A>G was associated with aberrant DNA methylation in offspring (OR 4.59, 95% CI 1.21-17.93). To the best of our knowledge, this is the first report of an association between maternal genotype and offspring methylation in pediatric ALL.

overall_design: Comparison of autosomal DNA methylation profiles using MCA-seq in children with ALL (N=51) and healthy bone marrow donors (N=6). Case-case comparisons of autosomal DNA methylation profiles according to 11 maternal variants in the methionine synthase gene.

first: Teresa

middle: T

last: Yiu

first: Philip

middle: J

last: Lupo

first: Spiridon

last: Tsavachidis

first: Rachel

last: Rau

first: Melissa

middle: L

last: Bondy

first: Karen

middle: R

last: Rabin

first: Lanlan

last: Shen

first: Michael

last: Scheurer


title: Quantitative Analysis of Wildtype and Neurog2CKO Heterozygote and Mutant Retinal Transcriptomes by RNA Sequencing

city: Davis

first: Nadean

last: Brown

address: 4407 Tupper Hall

department: Dept of Cell Biology and Human Anatomy

institute: University of California, Davis

summary: To generate an unbiased view of changes to the retinal gene network in Neurog2 retinal mutants, we generated and compared the P2 transcriptomes from control, heterozygote and mutant mice.  A pair of P2 retinas from each biologic replicate were used to produce libraries for high throughput sequencing (n = 5 biologic replicates/genotype).  Reads were aligned with BWA and Bowtie programs to the mm10 genome.  Aligned reads were then analyzed for differentially expressed transcripts using the CuffDiff program in the Galaxy online bioinformatics package (www.usegalaxy.org).

overall_design: Total RNA from Neurog2CKO/CKO(wildtype; n = 5), Chx10Cre;Neurog2CKO/+(heterozygote; n = 5), and Chx10Cre;Neurog2CKO/CKO(mutant; n = 5) P2 retinas.

first: Angelica

last: Kowalchuk


title: Mitochondrial dsRNA triggers antiviral signalling in humans

first: Nicholas

last: Proudfoot

summary: Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts capable of forming long double-stranded RNA structures. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here, we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single cell level and identify keyroles for the degradosome components, mitochondrial dsRNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to  the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms evolved to protect vertebrates against microbial and viral attack.

overall_design: We performed J2 IP dsRNA-seq from HeLa cell lysates by immunoprecipitation with anti-dsRNA (J2) mab. Untreated and siCntrl treated HeLa cells were used in the experiment.  Experiment was performed in duplicates from HeLa cells treated with siSUV3 and siPNPase.  Input RNA samples were ribodepleted with Ribo-Zero rRNA-removal kit. IPed RNA samples were not ribodepleted.

first: Ashish

last: Dhir

first: Somdutta


title: ZNF341 controls STAT3 expression and thereby immunocompetence

city: Freiburg

first: Claudia

last: Bossen

address: Breisacher Strasse 115

department: Center for Chronic Immunodeficiency

institute: University Hospital Freiburg (D)

summary: Signal-transducer-and-activator-of-transcription-3 (STAT3) is a central regulator of immune homeostasis. STAT3 levels are strictly controlled and STAT3 impairment contributes to several diseases including the monogenic autosomal-dominant hyper-IgE syndrome (AD-HIES). We investigated patients of four consanguineous families with an autosomal-recessive disorder resembling the phenotype of AD-HIES, with symptoms of immunodeficiency, recurrent infections, skeletal abnormalities, and elevated IgE. Patients presented with reduced STAT3 expression and diminished Th17 cell numbers, in absence of STAT3 mutations. We identified homozygous nonsense mutations in ZNF341, encoding a zinc-finger transcription factor. Wildtype-ZNF341 bound to and activated the STAT3 promoter, whereas the mutant variants showed impaired transcriptional activation, partly due to nuclear translocation failure. In summary, nonsense mutations in ZNF341 account for the STAT3-like phenotype in four autosomal-recessive kindred. Thus, ZNF341 is a previously unrecognized regulator of immune homeostasis.

overall_design: To determine binding sites of ZNF341 genome-wide, we performed ChIP against ZNF341 using 2 different antibodies and Input DNA as control.

first: Julia

last: Hartberger

first: Stefanie

last: Frey-Jakobs

first: Manfred

last: Fliegauf

first: Magdalena

last: Wehmeyer

first: Johanna

last: Neubauer

first: Vivien

last: Béziat

first: Anne

last: Puel

first: Jean-Laurent

last: Casanova

first: Bodo

last: Grimbacher


title: Pluripotent stem cell model of Nakajo-Nishimura syndrome untangles proinflammatory pathways mediated by oxidative stress

city: Kyoto

first: Akira

last: Niwa

address: 53, Shogoin-Kawahara-cho, Sakyo-ku

department: Department of Clinical Application

country: Japan

institute: CiRA, Kyoto University

summary: Nakajo-Nishimura syndrome (NNS) is an immunoproteasome-associated autoinflammatory disorder caused by a mutation of the PSMB8 gene. Although dysfunction of the immunoproteasome causes various cellular stresses attributed to the overproduction of inflammatory cytokines and chemokines in NNS, the underlying mechanisms of the autoinflammation are still largely unknown. To investigate and understand the mechanisms and signal pathways in NNS, we established a panel of isogenic pluripotent stem cell (PSC) lines with PSMB8 mutation. Activity of the immunoproteasome in PSMB8-mutant PSC-derived myeloid cell lines (MT-MLs) was reduced even without stimulation compared to non-mutant-MLs. Additionally, MT-MLs showed an overproduction of inflammatory cytokines and chemokines, with elevated reactive oxygen species (ROS) and phosphorylated p38 MAPK levels. Treatment with p38 MAPK inhibitor and anti-oxidants decreased the abnormal production of cytokines and chemokines. The current PSC model revealed a specific ROS-mediated inflammatory pathway, providing a platform for the discovery of alternative therapeutic options for NNS and related immunoproteasome disorders.

overall_design: RNA-sequencing was conducted to identify the genes expressed in reponse to stimulation in different manners between WT and MT cells

first: Fumiko

last: Honda-Ozaki

first: Madoka

last: Terashima

first: Megumu

last: Saito


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and TWIST1 knock out U87 xenograft mice transcriptomes

first: LEI

last: HUANG

department: Systems medicine and Bioengineering

institute: Houston Methodist Research Institute

summary: Purpose:Next-generation sequencing has revolutionized sytems-level celluar pathway analysis. The goals of this study are to compare the U87 cell xenograft GBM mice (U87 cell line) to TWIST1 knock out U87 cell xenograft GBM mice (TWIST1 knock out U87 cell line) using their transcriptomes

overall_design: Methods: Investigation of TWIST1 expression on glioblastoma malignancy in vitro and in vivo.

first: Lei

last: Huang

first: A

last: Mikheev


title: Cooptation of tandem DNA repeats for the control of epithelial-to-mesenchymal transition [miRNA-Seq]

first: Chiara

last: Balestrieri

overall_design: Total RNA from human pancreatic ductal adenocarcinoma cell lines was processed for multiparallel sequencing. Experiments were carried out in unmodified original cell lines (CAPAN-1, CAPAN-2, CFPAC-1, MiaPaCa-2, PANC-1 and PT45P1).

first: Gioacchino

last: Natoli


title: RNAseq skeletal muscle FUNDC1-mKO mice

city: Nanjing

first: Zhenji

last: Gan

address: 12 Xuefu Road

department: Model Animal Research Center of Nanjing University

institute: Nanjing University

summary:
This experiment was conducted to identify mRNA transcripts alteration in muscle from skeletal muscle-sepcific Fundc1-knockout mice. The following abstract from the submitted manuscript describes the major findings of this work.

Mitophagy directs muscle-adipose crosstalk to alleviate dietary obesity. Tingting Fu, Zhisheng Xu, Lin Liu, Qiqi Guo, Hao Wu, Xijun Liang, Danxia Zhou, Liwei Xiao, Lei Liu, Yong Liu, Min-Sheng Zhu, Quan Chen and Zhenji Gan.
The quality of mitochondria in skeletal muscle is essential for maintaining metabolic homeostasis during adaptive stress responses. However, the precise control mechanism of muscle mitochondrial quality and its physiological impacts remain unclear. Here, we demonstrate that FUNDC1, a mediator of mitophagy, plays a critical role in controlling muscle mitochondrial quality as well as metabolic homeostasis. Skeletal muscle-specific ablation of FUNDC1 in mice resulted in LC3-mediated mitophagy defect, leading to impaired mitochondrial energetics. This caused decreased muscle fat utilization and endurance capacity during exercise. Interestingly, mice lacking muscle FUNDC1 were protected against high-fat diet-induced obesity with improved systemic insulin sensitivity and glucose tolerance despite reduced muscle mitochondrial energetics. Mechanistically, FUNDC1 deficiency elicited a retrograde response in muscle that upregulated FGF21 expression, thereby promoting the thermogenic remodeling of adipose tissue. Thus, these findings reveal a pivotal role of FUNDC1-dependent mitochondrial quality-control in mediating the muscle-adipose dialogue to regulate systemic metabolism.

overall_design: Gastrocnemius muscle mRNA profiles of 8-week-old WT and FUNDC1-mKO mice were generated by deep sequencing, in replicate, using Illumina HiSeq 4000.

first: Tingting

last: Fu

first: Zhisheng

last: Xu


title: RNA-seq of cancer cell lines treated with T-025

city: Fujisawa

first: Kenichi

last: Iwai

state: Kanagawa

address: 26-1, Muraoka-Higashi 2-chome

institute: TAKEDA pharaceutical comapny

summary:
4 cancer cell lines were treated with T-025 for 6 hours
T-025 is a novel small molecule inhibitor of CLK.  CLK is a kinase that regulates pre-mRNA splicing.

overall_design: RNAs from NCI-H1048, MDA-MB-468, 786-O, or COLO320HSR cells treated with T-025 at 30, 100, 300 nM for 6 hours were subjected to deep sequencing using HiSeq2000

first: Misato

last: Kaishima


title: An epigenetic mechanism for cavefish eye degeneration

first: Aniket

last: Gore

address: Bldg 6B center drive

institute: National Institute of child health and human development

summary: Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution.  However, less is known about the role of epigenetics in this process.  Although previous studies have identified eye development genes associated with the loss of eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus1-6, no inactivating mutations have been found in any of these genes2,3,7-10.  Here we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave Astyanax mexicanus.  By performing parallel analyses in Astyanax mexicanus cave and surface morphs and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development.  The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.

overall_design: RNA-seq and WGBS on isolated cave and surface morphs of Astyanax mexicanus

middle: V

first: Brant

last: Weinstein


title: RNA-seq of MYC-inducible SK-MEL-28 cells

summary: MYC-inducible SK-MEL-28 cells with or without MYC-induction were analyzed

overall_design: RNAs from SK-MEL-28 vector or SK-MEL-28 Myc treated with or without Doxycycline for 54 hours were subjected to deep sequencing using HiSeq4000


title: Impact of Lmo2 expression on cell lineage decision during T-cell leukemogenesis [RNA-seq]

city: Salamanca

first: Isidro

last: Sánchez García

address: Campus Miguel de Unamuno

department: Centro de inv. del Cáncer (CIC).

institute: Inst. de Inv. Biomédica de Salamanca (IBSAL). H. U. de Salamanca. Inst. de Biología Molecular y Celular del Cáncer (IBMCC).

summary:
The goal of this study is compare the transcriptome profilling of different genetically engineered mouse models to the Human scenerio, in order to better understand the role of Lmo2 expression on cell lineage decision during T-cell leukemogenesis.
Results: The up-regulated Rosa26-Lmo2+Sca1-Cre and Sca1-Lmo2 signatures shows a significant enrichment in the human T-ALL group.
Conclusion: The similarity between Rosa26-Lmo2+Sca1-Cre and Human T-ALL signature is in accordance to the Human T-ALL situation wherein the expression of LMO2 is present throughout in tumor cells.

overall_design: 14 samples were analyzed representing different mouse model employed in the study,  including 4 control samples obtained from healthy thymus.

first: Idoia

last: García-Ramírez

first: Sanil

last: Bhatia

first: Guillermo

last: Rodríguez-Hernández

first: Inés

last: González-Herrero

first: Carolin

last: Walter

first: Sara

last: González de Tena-Dávila

first: Salma

last: Parvin

first: Oskar

last: Haas

first: Wilhelm

last: Woessmann

first: Martin

last: Stanulla

last: Schrappe

last: Dugas

first: Yasodha

last: Natkunam

first: Alberto

last: Orfao

first: Verónica

last: Domínguez

first: Belén

last: Pintado

first: Oscar

last: Blanco

first: Diego

last: Alonso-López

first: Javier

last: De Las Rivas

last: Martín-Lorenzo

first: Rafael

last: Jiménez

first: Francisco

last: García Criado

first: María

last: García Cenador

first: Izidore

last: Lossos

first: Carolina

last: Vicente-Dueñas

first: Arndt

last: Borkhardt

first: Julia

last: Hauer

last: Sánchez-García


title: Comprehensive analysis of hippocampal miRNAomes in humans and mice.

city: Beijing

first: Wei

last: Chen

state: Beijing

address: No. 95 Yong’an road, Xicheng District

department: Beijing Friendship Hospital

institute: Capital Medical University

summary: MicroRNAs (miRNAs) are small endogenous regulatory RNAs involved in hippocampal functions and lesions. Mouse has been regarded as an effective tool to explore the roles of miRNAs in hippocampus. Although the anatomical structure and physiological function across human and mouse hippocampi are highly conserved, the similarity and difference of hippocampal miRNAomes between the two species have not been well characterized. Our present study represents the first attempt to perform a systematic comparison of the miRNAomes between healthy human and mouse hippocampi using high-throughput sequencing followed by bioinformatic analyses. Contrasting between human and mouse hippocampal miRNAs shows a global similarity of expression pattern; however, the interspecific expression conservation of hippocampal miRNAs is positively associated with the interspecific sequence conservation. Although the type of the abundantly expressed hippocampal miRNAs is almost the same across the two species, only the enriched miRNAs in human hippocampus exhibit potential neuro-related activities. In addition, we also identified a novel miRNA and a species-specific miRNA in human hippocampus whose putative targets are associated with neuro-related activities. Collectively, our results can provide a framework to explore the ability of mice to model the physiological and pathological processes of human hippocampus and also aid in understanding the basis for higher order abilities of learning and memory at the miRNA level.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Total RNA from frozen hippocampi (7 humans and 7 mice) was purified using Trizol (Invitrogen, USA) protocol with no modifications. The concentration, purity and integrity were detected by Qubit 2.0 (Life Technologies, USA), Nanodrop (Thermo Scientific, USA), and Agilent 2100 bioanalyzer (Agilent Technologies, USA), respectively. One μg RNA was used to generate a small RNA sequencing library based on TruSeq Small RNA Sample Prep Kit version 2 (Illumina, USA) according to the manufacturer’s instructions. In brief, T4 RNA ligase was used to ligate RNA 5’ and 3’ adaptors to 5' and 3' ends of RNA, respectively. Adapter-ligated RNA was reverse-transcribed using a RNA RT Primer and the resulting cDNA was amplified in an 11-cycle PCR that used RP1 (RNA PCR Primer) and indexed RP1 primers. PCR products of 140–160 bp were isolated following electrophoresis through a 6% Novex Tris-borate polyacrylamide gel (Life Technologies, USA). Quality of the generated small RNA sequencing library were confirmed using Agilent 2100 bioanalyzer (Agilent Technologies, USA). High-throughput sequencing was carried out based on an Illumina HiSeq2500 system.

first: Chuan

last: Qin


title: LEC-induced gene expression changes in melanoma cells

city: Helsinki

first: Paivi

middle: Marjut

last: Ojala

address: Haartmaninkatu 8 (P.O.Box 63)

department: Research Programs Unit

country: Finland

institute: University of Helsinki

summary: RNA sequencing to analyze the gene expression changes in the WM852 and Bowes melanoma cells upon their co-culture with lymphatic endothelial cells (LECs). Platforms: NextSeq500 sequencer (Illumina) as quadruplicates

overall_design: There are n=3 of 1. monotypic WM852, 2. LEC co-cultured WM852, 3. monotypic Bowes, 4. LEC co-cultured Bowes

first: Pirita

last: Pekkonen

first: Giuseppe

last: Balistreri

first: Otso

last: Niiranen


title: Polyol pathway links glucose metabolism to the aggressiveness of cancer cells

city: Erlangen

first: Arif

last: Ekici

address: Schwabachanlage 10

institute: Institute of Human Genetics

summary: Cancer cells alter their metabolism to support their malignant properties. By transcriptomic analysis we identified the glucose-transforming polyol pathway (PP) gene aldo-keto-reductase-1-member-B1 (AKR1B1) as strongly correlated with epithelial-to-mesenchymal transition (EMT). This association was confirmed staining samples from lung cancer patients and from an EMT-driven colon cancer mouse model with p53 deletion. In vitro, mesenchymal-like cancer cells showed increased AKR1B1 levels and AKR1B1 knockdown was sufficient to revert EMT. An equivalent level of EMT suppression was measured by targeting the downstream enzyme sorbitol-dehydrogenase (SORD), further pointing at the involvement of the PP. Comparative RNA sequencing profiling confirmed a profound alteration of EMT in PP-deficient cells, revealing a strong repression of TGF-Beta signature genes. Mechanistically, excess glucose was found to promote EMT through autocrine TGF-Beta stimulation, while PP-deficient cells were refractory to glucose-induced EMT. PP represents a molecular link between glucose metabolism and cancer differentiation and aggressiveness, and a novel potential therapeutic target.

overall_design: 3x3 biological replicated samples; 2 groups of samples with shRNA-mediated specific gene inhibition and scrambled control cells

first: Paolo

last: Ceppi


title: SPT6 interacts with NSD2 and facilitates interferon-stimulated transcription

first: Razvan

middle: V.

last: Chereji

state: Maryland

address: 6 Center Drive, Room 2A14

department: NICHD

institute: National Institutes of Health

summary: SPT6, encoded by the SUPT6H in humans and Supt6 in mice, respectively, is a conserved histone chaperone that interacts with RNA polymerase II and participates in transcription elongation. However, the question of how SPT6 comes into play in transcriptional activation upon signaling, particularly in mammalian cells, has remained elusive. We investigated the contribution of SPT6 to interferon beta (IFNbeta) induced transcription in mouse NIH3T3 cells. IFNbeta triggers rapid and high level transcription of many IFN-stimulated genes (ISGs). We report here that SPT6 is recruited to ISGs after IFN stimulation. This recruitment was dependent on the interaction with the methyltransferase, NSD2. Further, siRNA-based SPT6 knockdown reduced levels of ISG activation. RNA-Seq analysis showed that SPT6 knockdown diminished about 50% of ISGs whose induction levels were higher than those unaffected by SPT6 knockdown. Under the tested conditions, SPT6 knockdown did not measurably change expression of constitutively expressed genes. This report highlights that SPT6 is recruited in a stimulus-dependent manner and elicits a major impact on signal induced transcription.

overall_design: RNA-seq of NIH3T3 cells.

first: Ryota

last: Ouda

first: Naoyuki

last: Sarai

first: Mira

last: Patel

first: Maxime

last: Debrosse

first: Vishal

last: Nehru

first: Mahesh

last: Bachu

first: David

last: Clark

first: Keiko

last: Ozato


title: Simultaneous methylation-level assessment of hundreds of CpG sites by targeted bisulfite PCR sequencing (TBPseq)

city: Daejeon

first: Kyuheum

last: Jeon

address: 125 Gwahak-ro

country: South Korea

institute: KRIBB

summary: Methylated-DNA sequencing technologies are producing vast amounts of methylome data from cancer samples, from which cancer-associated differentially methylated CpG sites (cDMCs) are continuously identified and filed. The inclusion of as many cDMCs as possible helps improve the accuracy of cancer diagnosis and sometimes identify cancer subtypes. However, the lack of an established method for the analysis of hundreds of cDMCs practically impedes their robust use in clinical medicine. Here, we tested the availability of targeted bisulfite-PCR-sequencing (TBPseq) technology for the assessment of methylation levels of a myriad of CpGs scattered over the genome. In randomly selected 46 cancer cell lines, multiplexed PCR yielded a variety of amplicons harboring 250 CpGs residing at promoters of 97 cancer-associated genes, all of which were sequenced in the same flow cell. Clustering analysis of the TBPseq-assessed methylation levels of target CpGs showed that the lung and liver cancer cell lines correlated relatively strongly with each other while they weakly correlated with colon cancer cells. CpGs at the MLH1 gene promoter, which are known to be hypermethylated in colon cancers, indeed were heavily methylated in the tested colon cancer cells. LIFR CpGs, also known to be methylated in colon cancers, were hypermethylated in the colon cancer cells. Moreover, MLH1 and LIFR promoter hypermethylations were found in colon cancer cells only, but not in biliary tract, liver, lung and stomach cancers cell lines. A meta-analysis with public cancer methylome datasets verified the colorectal cancer specificity of MLH1 and LIFR promoter methylation. These results demonstrate that our TBPseq-based methylation assessment could be considered an effective, accurate, and competitive method to simultaneously examine a large number of target cDMCs and patient samples.

overall_design: In randomly selected 46 cancer cell lines, multiplexed PCR yielded a variety of amplicons harboring 250 CpGs residing at promoters of 97 cancer-associated genes.

first: Byungkuk

last: Min

first: Jung

last: Park

first: Yong-Kook

last: Kang


title: EWSR1 influences alternative splicing through direct and indirect mechanisms

city: San Francisco

first: Marcus

last: Breese

address: 1550 Fourth St, 382

institute: UCSF

summary: FET family RNA binding proteins are implicated in cancer and neurological disorders and yet their biological function is incompletely understood. We used cross-linking and immunoprecipitation followed by high-throughput sequencing (HITS-CLIP) as well as gene expression and alternative splicing analysis by RNAseq to characterize the molecular function of the FET family RNA binding protein EWSR1. In HeLA cells, EWSR1 binding is enriched at 3’UTRs and at both 5’ exon/intron and 3’ intron/exon boundaries.  RNAs bound by EWSR1 are functionally enriched for RNA binding and RNA processing genes, suggesting a key role for EWSR1 in RNA regulation. While EWSR1 knock-down altered expression of hundreds of genes, only a subset of the transcripts for these genes were directly bound by EWSR1. By overlapping the HITS-CLIP data with the RNAseq splicing analysis we identified genes where EWSR1 binding is more likely to directly affect splicing.  EWSR1 regulated splicing events were varied and included exon inclusion, alternative 3 exon usage and intron retention. These  results indicate that EWSR1 has pleiotropic effects on splicing and that these effects are caused both by direct regulation of splicing and through effects on a large number of RNAs that themselves code for splicing effectors. Further understanding of this complex splicing regulation network may be important to disentangle the role of EWSR1 in multiple human diseases.

overall_design: CLIP: 2 replicates; RNAseq: 3 Controls (GFP siRNA), 4 Experimental (EWSR1 siRNA)

first: E

last: Sweet-Cordero


title: Genome-wide maps of H3 and H2A.Z RITE ChIP-sequencing

city: Amsterdam

first: Tessy

last: Korthout

state: Noord-Holland

address: Plesmanlaan 121

department: Gene Regulation

country: Netherlands

institute: The Netherlands Cancer Institute

summary: We performed ChIP-sequencing for both H2A.Z and H3 RITE after 4 hours in G1-arrest. In addition, we report H2A.Z and H3 ChIP-sequencing data for samples of replicating cells.

overall_design: We compare the V5 (old) and HA (new) IP's for both histone marks with the canonical pattern in log phase.

first: Marit

last: Terweij


title: HIV-1 perturbs homeostatic ILCs, unmasks ILC1 plasticity, and boosts TCF7+ memory NK cells

first: Alan

middle: G

last: Derr

state: Massachusetts

address: 55 Lake Ave North

department: Program in Bioinformatics and Integrative Biology

institute: University of Massachusetts Medical School

summary: Bulk RNA-seq, and single cell RNA-Seq using inDrops methodology, were used to compare two populations of cells that had been sorted by flow cytometry from human peripheral blood mononuclear cells (PBMCs) from two donors, G27 and G33. To sort the cells, PBMCs were stained with antibodies to 11 lineage markers (FITC), anti-CD56 (PE), and anti-CD94 (APC). The 11 lineage marker positive cells were excluded to identify the innate lymphocyte cell (ILC) population. Then we sorted the Lin-CD56+CD94- cells (CD127-ILC1s, 'neg') from the Lin-CD56+CD94+ cells (NK cells, 'pos') with a BD FACSAria IIu. Libraries were generated from each of these two populations.

overall_design: Quantification and analysis of FACS-sorted human ILC cells from two donors.

first: Yetao

last: Wang

first: Kyle

last: Gellatly

first: Smita

last: Jaiswal

first: Alper

last: Kucukural

first: Patrick

last: McDonel

first: Thomas

last: Greenough

first: JeanMarie

last: Houghton

first: Manuel

last: Garber

first: Jeremy

last: Luban


title: UTX-mediated enhancer and chromatin remodeling suppresses myeloid leukemogenesis through noncatalytic inverse regulation of ETS and GATA programs

city: Cambridge

first: Brian

last: Huntly

address: Hills Road

department: Department of Haematology

institute: University of Cambridge

summary: The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs.

overall_design: Refer to individual Series


city: Dundee

first: Dhaval

last: Varshney

address: Dow Street

department: Centre for Gene Regulation and Expression

institute: University of Dundee

summary: mRNA cap addition occurs early during RNA pol II transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription, independently of mRNA capping and translation. In cells, sub-lethal suppression of RNMT-RAM reduces RNA pol II occupancy, net mRNA synthesis and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independently of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA-binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription associated factors including RNA pol II subunits, SPT4, SPT6 and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2B K120 ubiquitination, H3 K4 and K36 methylation, RNA pol II S5 and S2 phosphorylation and PAFc recruitment. These findings suggest that multiple interactions between RNMT-RAM, RNA pol II factors and RNA along the transcription unit stimulate transcription.

overall_design: CLIP experiment performed on HA-RNMT expressing HeLa cells. RNASeq and ChIP-Seq performed on HeLa cells treated with siRNA for RAM and scrambled control.

first: Victoria

last: Cowling


title: Transcriptome analysis of N2 and flp-6 Caenorhabditis elegans at 25 °C

city: Taipei

first: Chun-Liang

last: Pan

address: No.7.Chung San South Road

department: Institute of Molecular Medicine

country: Taiwan

institute: National Taiwan University

summary: Temperature is a prominent environmental stimulus that influences life span. Previous studies indicate that in Caenorhabditis elegans, thermosensory perception in the AFD neuron maintains life span at warm temperatures. How thermosensation is translated into neuronal signals that shape aging remains elusive. We found that the Caenorhabditis elegans CREB crh-1, as well as several key genes in AFD thermosensory transduction, were specifically required for normal life span at warm temperatures. crh-1 acted in the AFD to increase transcription of the CRE-containing neuropeptide gene flp-6 in a temperature-dependent manner. Both crh-1 and flp-6 were necessary and sufficient for longevity at warm temperatures, and their effects depended on the AIY interneuron. Moreover, flp-6 signaling downregulated ins-7/insulin and several insulin pathway genes, whose activity compromised life span. We postulate that temperature experience is integrated in the thermosensory neurons to generate CREB-dependent neuropeptide signals that antagonize insulin signaling and promote temperature-specific longevity.

overall_design: total RNA profiles of L4 wild type (N2) and flp-6 mutant Caenorhabditis elegans were generated by deep sequencing using Illumina Solexa.


title: UTX-mediated chromatin remodeling suppresses myeloid leukaemogenesis through non-catalytic inverse regulation of ETS and GATA transcriptional programs (ChIP-Seq)

summary: The H3K27 lysine-specific demethylase UTX is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukaemogenesis through non-catalytic functions, a property it shares with its catalytically inactive Y-chromosome paralogue, UTY. In keeping with this, we demonstrate concomitant loss/mutation of UTX and UTY in multiple human cancers. Mechanistically, global genomic profiling revealed only minor changes in H3K27Me3, but significant and bidirectional alterations of H3K27Ac and chromatin accessibility, alterations in ETS and GATA factor binding and altered gene expression upon Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX coordination of ATP-dependent chromatin remodelling and competition between UTX and ETS factors for binding at critical DNA-sequences. Collectively, our findings reveal a dual role for UTX in suppressing acute myeloid leukaemia via inhibition of oncogenic ETS and upregulation of tumour-suppressive GATA programmes. We propose that equivalent changes in tissue-specific oncogenic and tumour-suppressive transcriptional programmes operate in other UTX-mutated cancers.

overall_design: ChIP-Seq analysis for two histone modifications (H3K27me3 and H3K27Ac) and ATAC-seq- comparison between mice deficient for the histone demethylase UTX (Utx-/-) vs wild type (Utx +/+).

first: George

last: Vassiliou


title: UTX-mediated chromatin remodeling suppresses myeloid leukaemogenesis through non-catalytic inverse regulation of ETS and GATA transcriptional programs (ATAC-Seq)


title: What happens to Prochlorococcus when the sun does not come up?

first: Steven

last: Biller

address: 15 Vassar St, MIT 48-424

department: Civil and Environmental Engineering

institute: MIT

summary: We examine how the transcriptome of Prochlorococcus strain NATL2A changes in response to extended light deprivation, both when grown alone and in the presence of a naturally co-occurring heterotroph, Alteromonas macleodii MIT1002.

sample_organism:
Alteromonas macleodii
Prochlorococcus marinus str. NATL2A

overall_design: Samples are collected over time from cultures experiencing either a 13:11 light:dark light cycle or that remain in the dark following the end of the expected night. Samples are collected from each of 3 biological replicate cultures of Prochlorococcus, grown either alone or in co-culture with a marine Alteromonas.

first: Allison

last: Coe

first: Sara

middle: E

last: Roggensack

first: Sallie

middle: W

last: Chisholm


title: Genome-wide mapping of the RNA targets of the Pseudomonas aeruginosa riboregulatory protein RsmN

city: Nottingham

first: Stephan

last: Heeb

state: Nottinghamshire

address: Centre for Biomolecular Sciences, University Park, University of Nottingham

institute: University of Nottingham

summary: Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In P. aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for rsmA or rsmAN mutants, probably as a consequence of different binding affinities. New targets for the Rsm system, previously masked by the presence of RsmN were identified. These include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of the two proteins to specific CrcZ target sequences is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues.

overall_design: To identify direct RsmN targets in P. aeruginosa and genes that are post-transcriptionally regulated by RsmN through interaction with their corresponding transcripts, RsmN was expressed in strain PAO1-L and co-purified with its binding RNAs. These were extracted and subjected to RNASeq to characterize the RsmN regulon under the growth conditions used. Total RNA was also extracted from the same culture samples to determine specific RNA abundances and to compare these to those of the RsmN-bound fractions as enrichment ratios.  Applying a stringent 3.5-fold minimum enrichment cut-off, a total of 503 transcripts was identified as transcripts bound by RsmN, several of which were experimentally validated. The Preliminary data set is of two conditions, total RNA and RsmN-bound RNA both in duplicate. The final study is of the same two conditions but in triplicate.

last: Romero

first: Hazel

last: Silistre

first: Laura

last: Lovelock

last: Wright

first: Kok-Gan

last: Chan

first: Kar-Wai

last: Hong

first: Paul

last: Williams

first: Miguel

last: Camara


title: RNA seq analysis of pathogen-induced transcriptome changes in tomato plants overexpressing the Arabidopsis ELP4 gene

city: GAINESVILLE

first: FAHONG

last: YU

state: FL

address: 2033 Mowry Rd

department: ICBR

institute: UNIVERSITY OF FLORIDA

summary: The goal of the RNA seq was to investigate the transcriptome changes induced by Pseudmonas syringae pv. tomato J4 in wild-type tomato 'Moneymaker' and transgenic 'Moneymaker' overexpressing the Arabidopsis ELP4 (AtELP4) gene. Results showed that P. syringae pv. tomato J4 induced dramatic transcriptional changes in both the wild-type and transgenic tomato plants.  Interestingly, a group of defense genes including PR-5x, Pti5, PR1b1, and CHI3/9/14/17, which are associated with resistance to the hemibiotrophic bacterial pathogen Ralstonia solanacearum, were induced to higher levels in the AtELP4 transgenic tomato than in the wild type at 8 and 24 hr after P. syringae pv. tomato J4 infection.  These results indicate that overexpression of AtELP4 in tomato leads to faster and/or stronger induction of some defense genes.

overall_design: Three replicates with leaves from 6 plants per genotype were collected at 0, 8, and 24 hr after P. syringae pv. tomato J4 infection. Leaf tissues were collected and subjected to total RNA extraction.  After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).  The RNA samples from the three biological replicates were pooled and equal amounts of RNA from the pooled samples were used for RNA seq analysis.

first: Juliana

last: Pereira

first: Yanping

last: Zhang

first: Fahong

last: Yu

first: Jeffrey

middle: B

last: Jones

first: Zhonglin

last: Mou


title: Loss of the Melanocortin 4 Receptor in mice causes dilated cardiomyopathy.

city: 2215 Garland Ave

first: Michael

last: Litt

state: TN

address: 702 Light Hall

department: MPB

institute: Vanderbilt University

summary: Deletion of the melanocortin-4 receptor in mice causes dilated cardiomyopathy that is independent of obesity.

overall_design: RNA from left ventricular tissue of MC4R-/- and WT was characterized using RNA-seq

first: Roger

middle: D

last: Cone


title: Epithelial-mesenchymal transition in cancer stem cells and during metastasis of a squamous cell carcinoma

status: Public on Apr 30 2018

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

country: Germany

institute: Universitaetsmedizin Goettingen

summary: Scientific background: Current understanding of carcinogenesis supposes a heterogeneous composition of malign tumors from cancer stem cells (CSCs) and stromal cells. CSCs most likely play an important role during treatment failure and metastasis. A possibly associated phenomenon is the phenotype of cancer cells undergoing an epithelial-mesenchymal transition (EMT). This study investigated the role of EMT in CSCs of a HPV-negative pharyngeal squamous cell carcinoma during metastasis. Methods: A cell line was established from a HPV-negative pharyngeal squamous cell carcinoma. Enrichment of cancer stem cells was performed by stem cell-enriching conditions in spheroid colonies. Gene expression profiling was performed (Human 8 × 60 K design array, Agilent Technologies) and the phenotype EMT was assessed by a gene set (MSigDB: M5930, Hallmark_epithelial_mesenchymal_transition) applying Gene set expression analysis (GSEA). A validation of the results was carried out by immunohistochemical staining and flow cytometry of CD44 und E-Cadherin. Results: Primary tumor and metastasis were highly positive for CD44. E-Cadherin was shown in primary tumor and the cell line derived from it. Flow cytometry showed a heterogeneous composition of the stem cell-enriched spheroid colonies with the appearance of a distinct population with the loss of E-Cadherin. GSEA showed significant enrichment of the EMT-phenotype in the primary tumor (FDR < 25 %, p < 5 %) compared to the other phenotypes. Conclusions: In this study, CSCs showed mesenchymal characteristics. EMT may be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active in CSCs during metastasis and at least partially turned back later.

overall_design: 6 different phenotypes were analysed, 2 of which (u45, H170) were for internal reference only. 4 phenotypes were included into further analyses (T, M, NZK, SphK) and consisted from 3 biological replicates each.

first: Friedrich

last: Ihler


title: MicroRNAs into body fluids from ten organs of mice exposed to cigarette smoke

city: Genoa

first: Alberto

last: Izzotti

address: Via A. Pastore 1

institute: University of Genoa

summary:
The purpose of the present study was to validate the application of this epigenetic biomarker by using less invasive collection procedures.Using microarray analyses, we measured 1135 microRNAs in 10 organs and 3 body fluids of mice that were either unexposed or exposed to mainstream cigarette smoke for up to 8 weeks. The results obtained with selected miRNAs were validated by qPCR
The lung was the main target effected by smoke (190 dysregulated miRNAs), followed by skeletal muscle (180), liver (138), blood serum (109), kidney (96), spleen (89), stomach (36), heart (33), bronchoalveolar lavage fluid (32), urine (27), urinary bladder (12), colon (5), and brain (0). Skeletal muscle, kidney, and lung were the most important sources of smoke-altered microRNAs in blood serum, urine, and bronchoalveolar lavage fluid, respectively

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Young mice were either untreated or exposed to MCS for up to 8 weeks.  RNA was purified from 10 organs and 3 biological fluids, and the expression of 1135 miRNAs was evaluated by microarrays.  For selected miRNAs, the results were validated by qPCR

first: Mariagrazia

last: Longobardi

first: Sebastiano

last: La Maestra

first: Rosanna

last: Micale

first: Alessandra

last: Pulliero

first: Anna

last: Camoirano

first: Marta

last: Geretto

first: Francesco

last: D'Agostini

first: Rumen

last: Balansky

first: Mark

last: Miller

first: Vernon

last: Steele

first: Silvio

last: De Flora


title: Transcriptome analysis of human embryonic stem cells (hESCs) upon siRNA-mediated knockdown of hESC-essential genes

city: Jerusalem

first: Mordecai

last: Peretz

address: The Edmond J. Safra Campus

institute: Hebrew University of Jerusalem

summary: The maintenance of pluripotency requires the coordinated expression of a set of essential genes. Thorough identification of these genes is vital for gaining a better understanding of the pluripotent state. To this end, we have recently generated a genome-wide loss-of-function library in haploid human pluripotent stem cells utilizing the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using nearly 180,000 single guide RNAs (sgRNAs) and targeting 18,166 protein-coding genes. By combining the results of our high-throughput genetic screening with the analysis of publicly available RNAseq data of pluripotent stem cells and several somatic cells, we have determined the essential genes which have an enriched expression in hESCs and termed these genes as hESC-essential genes. Here, we have individually knocked down four of these hESC-essential genes with siRNAs, namely SALL4, DSCC1, SEPHS1 and VRTN, in human embryonic stem cells and analyzed the transcriptomes of the cells upon downregulation of these transcripts in order to unravel part of the pathways affected by hESC-essential genes.

overall_design: For each of the gene knockdown conditions, three biological replicates were analyzed. hESCs were transfected with commercial pooled siRNAs targeting either of the four genes targeted along with the control siRNAs. Four control conditions included in the study were treated with two independent control siRNAs with two biological replicates for each.

first: Atilgan

last: Yilmaz

first: Nissim

last: Benvenisty