title: Locus-specific control of the de novo DNA methylation pathway [MethylC-seq]

status: Public on Apr 14 2018

city: San Diego

first: Julie

last: Law

state: CALIFORNIA

address: 10010 N Torrey Pines Rd, La Jolla

institute: Salk Institute for Biological Studies

summary: Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.

overall_design: 12 MethylC-seq libraries were sequenced

first: Ming

last: Zhou


title: Locus-specific control of the de novo DNA methylation pathway [mRNA-seq]

country: USA

type: Expression profiling by high throughput sequencing

overall_design: 22 mRNA-seq libraries were sequenced


title: Locus-specific control of the de novo DNA methylation pathway

type: Methylation profiling by high throughput sequencing

type: Non-coding RNA profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: A molecular fingerprint for terminal abdominal aortic aneurysm progression

status: Public on Feb 27 2018

city: Munich

first: Bernd

middle: Henrik

last: Northoff

address: Marchioninistr. 15

department: Institute of Laboratory Medicine

country: Germany

institute: Ludwig-Maximilians-University Munich

summary: Analysis of differential gene expression for rutured vs stable abdominal aortic aneurysms (AAA) and for intermediate size (≤55mm) vs large (>70mm) AAA.

overall_design: This observational study compares transcriptome-wide exression analysis of elective stable (n = 31) vs ruptured (n = 17) AAA and intermediate size (n = 15) vs large (n = 16) AAA.

middle: H


title: Mutating Zta (182N) changes sequence specific DNA binding (5mC + 5hmC)

status: Public on Jun 04 2019

city: Bethesda

first: Desiree

last: Tillo

state: MD

address: 41 Center Dr, Room D310

institute: NIH/NCI

summary: The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated DNA.  To study the contribution of the conserved N182 amino acid to sequence specific Zta DNA binding, we replaced it with five other amino acids:  serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V).  We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of double-stranded DNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)).  With unmethylated DNA, Zta(N182S) binds variants of the consensus TRE (TGA-G/C-TCA) motif, such as TGA-G/C-TGA and TCA-G/C-TGA where C at position 3 is replaced with G in one or both half sites of the motif.  Zta(N182S) also binds stronger to DNAs containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing GTAA with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C at position 3 is replaced with A.  Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined.  Zta(N182T) DNA binding is weaker to all types of DNA examined.  Our data highlight that mutation of ZtaN182 with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).

overall_design: Protein binding microarray (PBM) experiments were performed for the DNA binding domain of the Epstein Barr Virus Zta protein and for 5 mutant constructs of Zta in which the asparagine (N) at position 182 of wild type Zta was changed to serine (N182S), glutamine (N182Q), threonine (N182T), isoleucine (N182I), or valine (N182V).   Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded and methylated or unmethylated 44K Agilent microarrays, containing a DeBruijn sequence design, in order to determine their sequence preferences. Details of the methylated PBM protocol are described in Mann et al., Genome Research, 2013


title: Genome-wide sequencing and comparative profiling of cattle sperm DNA methylome

status: Public on Mar 08 2018

city: Wuhan

first: Yang

state: Hubei

address: Shizishanjie #1

department: College of animal science

country: China

institute: Huazhong Agricultural University

summary: Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperms through comparison with three bovine somatic tissues (mammary grand, brain and blood). Large differences between cattle sperms and somatic tissues were observed in the methylation patterns.

overall_design: Genomic DNA for each tissue was isolated according to the QIAamp DNA Mini Kit protocol (QIAGEN, Valencia, CA, USA). The quality of DNA samples was evaluated using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) including degradation, and potential RNA contamination, and purity (OD260/OD280), and concentration using a spectrophotometer (NanoDrop Technologies, Rockland, DE) to meet the requirements for library construction. The qualified genomic DNA from somatic tissues and sperm were used to construct a library. The library different lanes of a HiSeq X Ten (Illumina, San Diego, CA, USA) to generate 150-bp paired-end reads by Novogene (Novogene, Beijing, China).

first: George

middle: E

last: Liu


title: A transcriptome-wide divergence in protein translation scales with LIN28B expression

status: Public on Dec 13 2018

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

summary: We report the mapping of LIN28B binding to RNA in HEK293A at various LIN28B expression levels using enhanced CLIP. We provide small RNA sequencing data regarding miRNA expression in HEK293A and those miRNAs bound by AGO2. We also profile the effect of LIN28B expression on transcriptome-wide ribosome occupancy in HEK293A via RNA-seq. Finally, we provide orthogonal regulatory data by profiling the effect of LIN28B expression on transcriptome-wide ribosome occupancy in NIH3T3 via RNA-seq.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of LIN28B binding using enhanced CLIP via 2 biological replicates of RNA fragments derived from LIN28B immunoprecipitation and 2 control input replicates for 5 LIN28B expression conditions. X0 is lin28b knockout. X1 through X4 are increasing levels of lin28b expression (X1 < X2 < X3 < X4). X1 through X4 lie within the naturally occurring spectrum of lin28b expression from other cell types such as HepG2, NTera2 and iPSC. Examination of small RNAs expressed in HEK293A and those specifically bound by AGO2, in the presence or absence of LIN28B or transfected let-7a miRNA, single replicate. Examination of changes in transcriptome-wide ribosome occupancy in HEK293A via total RNA (2 replicates) and ribosome-bound RNA (2 replicates) for 5 different LIN28B expression conditions.  Experimental design for examining changes in transcriptome-wide ribosome occupancy in NIH3T3 were the same as for HEK293A.

first: Frederick

last: Tan

first: Shashank

last: Sathe

first: Emily

last: Wheeler

first: Julia

last: Nussbacher


title: Single cell RNA-seq profiles of primary, metastatic, drug-resistant and drug-holiday cells

status: Public on Nov 22 2018

city: Singapore

first: Yiqun Elaine

last: Cao

address: 8 College Road

country: Singapore

institute: Duke-NUS Medical School

summary: We employed Single cell RNA-seq to identify the transcriptome of cisplatin sensitive, resistant and drug-holiday cells from patient-derived OSCC cells isolated from primary and metastatic sites.

overall_design: Single-cell RNA-seq profiles of in sensitive and drug-resistant cells from OSCC

first: Ankur

last: Sharma

first: Ramanuj

last: DasGupta


title: Identification of an IL-1-induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR- PCa cells

status: Public on Feb 01 2018

city: Richardson

first: Nikki

last: Delk

address: 800 West Campbell Road

department: Biological Sciences

institute: University of Texas at Dallas

summary: In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells.  However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression.  In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum and promotes PCa bone metastasis.  IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable; suggesting that IL-1 may also contribute to AR-targeted therapy resistance.  Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells.  Taken together, we hypothesize that IL-1 reprograms AR positive (AR+) PCa cells into AR negative (AR-) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival in the inflammatory tumor microenvironment.  Thus, we employed RNA sequencing to identify pathways that are modulated by IL-1 concomitant with IL-1-induced AR repression in PCa cells.  Comparative analysis of sequencing data from the AR+ LNCaP PCa cell line versus the AR- PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells, and includes AR and AR target gene repression and the induction of prosurvival, lineage, and cancer stem cell genes.  Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival in an inflammatory tumor microenvironment.  Our data supports that IL-1 reprograms AR+ PCa cells to mimic AR- PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival.

overall_design: PCa cells were treated with vehicle control or IL-1β or grown in bone marrow stromal cell CM (conditioned media) from HS-5 bone marrow stromal cell lines for 3 days. At Day 3 RNA was extracted from 3 biological replicates for RNA-sequencing and confirmation analyses were performed using western blot and RT-QPCR.

first: Shayna

last: Thomas-Jardin

first: Mohammed

last: Kanchwala

first: Joan

last: Jacob

first: Sana

last: Merchant

first: Rachel

last: Meade

first: Nagham

last: Gahnim

first: Afshan

last: Nawas

first: Chao

last: Xing


title: Integrative genomic analysis in HCC samples [MYH]

status: Public on Jan 19 2018

city: Beijing

first: Gangqiao

last: Zhou

address: No. 27, Taiping Road, Haidian District

institute: Beijing Institute of Radiation Medicine

summary: We integrated the copy number data with gene expression data from the same HCC samples, and identified fifteen putative driver genes with recurrently genomic aberrations and their associated modules in HCC. We further confirmed empirically that three putative driver genes (MYH10, CEP104 and RRS1) play significant roles in tumor initiation and progression of HCC. Notably, we demonstrated that RRS1 regulates the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus in HCC. Altogether, these data provide insights into novel cancer driver genes and suggested molecular targets for treatment for HCC.

overall_design: Total RNA obtained from the human HepG2 cells with MYH10 shRNAs or HepG2 with control shRNA. Three biological replicates each.

first: Peiyao

last: Li

first: Yuqing

last: Han

first: Pengbo

first: Xia

last: Xia

first: Xi

last: Chen


title: The role of Polycomb Repressive Complex 2 (PRC2) in Schwann cell responses to peripheral nerve injury

status: Public on Jan 01 2018

city: Madison

first: John

last: Svaren

state: WI

address: 1500 Highland Ave.

department: Comparative Biosciences

institute: University of Wisconsin

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus


title: Integrative genomic analysis in HCC samples

type:
Expression profiling by array
Genome variation profiling by SNP array
SNP genotyping by SNP array


title: Oryza sativa Argonaute 18 is a regulator of male sterility (RNA-Seq)

status: Public on Jan 15 2018

city: Bangalore

first: Shivaprasad

last: Padubidri V.

address: GKVK Campus, Bellary road

department: Plant epigenetics

country: India

institute: National Center for Biological Sciences

summary: Artificial miRNA mediated knock-down ago18 transgenic lines [ago18(1), ago18(2)] showed developmentally compromised phenotype in both vegetative and reproductive tissues compared to wild-type control. We have performed RNA seq to investigate underlying molecular mechanism and to reveal functional role of AGO18 in rice.

overall_design: We have performed RNA seq analysis using vegetative and reproductive tissues of both WT and knock-down tansgenic lines. Four weeks old seedlings were considered as vegetative tissues and two different stage of panicle were examined as reprodictive tissues. Libraries were prepared from total RNA isolated from the tissues mentioned.

first: Padubidri

middle: V

last: Shivaprasad

first: Swetha

last: Chenna

first: Soumita

last: Das


title: Id2-deficient NK cells acquire a naïve-like fate (Affymetrix data set)

status: Public on Feb 26 2018

city: Chicago

first: Barbara

middle: L

last: Kee

state: Illinois

address: 924 E57th st

department: Pathology

institute: The University of Chicago

summary: All innate lymphoid cells (ILC) constitutively express and require the small helix-loop-helix protein ID2 but the functions of ID2 are not well understood in these cells. Here we show that natural killer (NK) cells, the prototypic ILC, can develop in the absence of ID2 but lose their innate properties and remain as CD27+CD11b- cells that fail to mature into cytotoxic effectors. We show that ID2 broadly limited chromatin accessibility at E protein binding sites near T lymphocyte-associated genes including multiple chemokine receptors, cytokine receptors, and signaling molecules. Moreover, ID2 prevented the conversion of CD27+CD11b- NK cells from a CD8 memory precursor-like chromatin accessibility state toward a naïve-like chromatin accessibility state, and altered their functional capacity. Finally, we demonstrate that increased expression of the naïve T cell-associated helix-loop-helix protein ID3 was required for development of ID2-deficient NK cells, indicating that completely unfettered E protein function is incompatible with NK cell development. These data solidify the roles of ID2 and ID3 as mediators of effector and naïve gene programs, respectively, and revealed a critical role for ID2 in promoting a chromatin state and transcriptional program in CD27+CD11b- NK cells that supports the innate properties of these cells and their ability to undergo cytotoxic effector differentiation.

overall_design: CD19-Ter119-CD122+NK1.1+CD27+CD11b- natural killer cells were sorted from the bone marrow of 3 Rag1-/-Id2f/f and 3 Rag1-/- GzmbCre Id2f/f mice (littermates) and RNA was extracted for analysis by Affymetrix microarray.

first: Erin

middle: C

last: Zook

first: Zhong

middle: Y

first: Yiying

last: Xu

first: Rene

middle: F

last: de Pooter

first: M

last: Verykokakis

first: Aimee

last: Beaulieu

first: Anna

last: Lasorella

first: Mark

last: Maienschein-Cline

first: Joseph

last: Sun

first: Mikael

last: Sigvardsson


title: Histone methylation in rat peripheral nerve

summary: The goal of this study was to determine the distribution of H3K27me3 in rat peripheral nerve, which is formed by PRC2, polycomb repressive complex 2.

overall_design: H3K27me3 ChIP samples were prepared from rat sciatic nerve.

first: Ki

last: Ma


title: A TAD boundary at the HoxD locus segregates opposing limb regulatory landscapes and their target genes

status: Public on Dec 22 2017

city: Geneva

first: Eddie

last: Rodríguez-Carballo

address: 4, Boulevard d'Yvoy

department: Department of Genetics and Evolution

country: Switzerland

institute: Université de Genève


title: Transcriptomic comparison of E13.5 Primordial Germ Cells (PGCs) from Fancg-/- and WT mice

status: Public on Nov 30 2017

city: Fontenay aux roses

first: Pierre

last: Fouchet

address: 10, route du panorama

country: France

institute: CEA

summary: Microarray analysis was performed in order to detail the global changes of gene expression in  Fancg -/- embryonic  Primordial Germ Cells and decipher molecular pathways underlying defects of primordial germ cell development in KO embryos.

overall_design: PGC from  E13.5 male embryos were sorted by flow cytometry, RNA exracted and used for Affymetrix microarray analysis. Three individual samples of 10,000 PGCs (pooled from at least five embryos for each sample) were processed for WT and KO genotypes

first: Amandine

last: Jarysta

first: Lydia

last: Riou


title: A non-canonical GLTSCR1L-containing BAF complex mediates H3K27ac-dependent enhancer activities

status: Public on Oct 17 2018

city: Copenhagen

first: Robin

last: Andersson

address: Ole Maaloes Vej 5

department: Biology

country: Denmark

institute: University of Copenhagen

summary: H3K27ac is known to associate with regulatory active enhancers, but it’s exact role in enhancer function remains elusive. Using mass spectrometry-based interaction proteomics, we identified the Super Elongation Complex (SEC) and GBAF, a non-canonical GLTSCR1L- and BRD9-containing SWI/SNF chromatin remodeling complex, to be major interactors of H3K27ac. Our interaction proteomics analysis refined the composition of GBAF by adding BCL7A/B/C as a new subunit. We further characterized the conserved GLTSCR1/1L-contained “GiBAF”-domain, which we found to be responsible for GBAF complex formation and nuclear localization. Inhibition of the bromodomain of BRD9 revealed interaction between GLTSCR1L and BRD9 to be H3K27ac dependent and led to GLTSCR1L dislocation from its preferred binding sites at H3K27ac-associated enhancers. GLTSCR1L disassociation from chromatin resulted in a genome-wide downregulation of enhancer transcription. Our results indicate that GBAF is an enhancer-associated chromatin remodeller important for the correct transcriptional and regulatory activity of enhancers.

overall_design: CAGE-seq (with 3 replicates) and ATAC-seq profiling of HeLa S3 cells untreated (DMSO) and treated with bromodomain-containing protein 9 inhibitor (I-BRD9) on a EGF-induced time course (0,30,60 and 240 minutes).  ChIP-seq for BRD9 and GLTSCR1L proteins before (DMSO) and after treatment (I-BRD9) for time point 0 and corresponding inputs.

first: Michiel

last: Vermeulen


title: CITE-seq: Large scale simultaneous measuremnt of epitopes and transcriptomes in single cells

status: Public on Jul 31 2017

city: New York

first: Marlon

last: Stoeckius

state: New York

address: 101 Avenue of the Americas

department: Innovation Lab

institute: New York Genome Center

summary: Recent high-throughput single-cell sequencing approaches have been transformative for understanding complex cell populations, but are unable to provide additional phenotypic information, such as protein levels of cell-surface markers. Using oligonucleotide-labeled antibodies, we integrate measurements of cellular proteins and transcriptomes into an efficient, sequencing-based readout of single cells. This method is compatible with existing single-cell sequencing approaches and will readily scale as the throughput of these methods increase.

overall_design: Cord blood mononuclear cells were profiled by CITE-seq using a panel of 13 antibodies.


title: UMCU colon cancer - Biopsies Of Surgical Specimens (BOSS)

status: Public on Aug 01 2017

city: Amsterdam

first: Richard

last: Volckmann

state: Noord-Holland

address: van der Boechorststraat 6a

department: Laboratory of Experimental Oncology and Radiobiology

country: Netherlands

institute: msterdam UMC and University of Amsterdam

summary: Validation of the RT-qPCR test by comparison with the 4-gene microarray test and the original random forest-based CMS (consensus molecular subtypes) classifier

overall_design:
For validation of the RT-qPCR test and assessment of intratumor heterogeneity, 20 colon cancer resection specimens were collected from routine surgery. Immediately after resection, five biopsy samples from randomly selected non-adjacent tumor areas were obtained with endoscopic biopsy forceps to simulate the clinical diagnostic setting. 29 samples from 7/20 specimens, representative of the encountered intra-tumoral variation in RT-qPCR-based predicted chances of CMS4 in the entire series, were selected for full Affymetrix gene expression profiling. This allowed direct comparison of the RT-qPCR test with the 4-gene microarray test and the original CMS classifier.
Please note that  [1] CMS classification for each sample was performed by combining various datasets, including the one uploaded here. If other datasets are combined, or if solely this dataset is analyzed, CMS classification could vary slightly, due to required post-processing steps. Therefore the classification information was not included as a sample characteristic. Users that wish to review the data can classify the samples as described in the CMS paper (Guinney et al. Nat Med 2015).
[2] From each tumor multiple biopsies were obtained from different regions; BOSS+number represent specimen ID, the roman numeral represents the biopsy number

first: I

last: Ubink

first: S

middle: G

last: Elias

first: C

middle: B

last: Moelans

middle: M

last: Laclé

first: W

last: van Grevenstein

first: P

middle: J

last: van Diest

last: Borel Rinkes

first: O

last: Kranenburg


title: Gene expression of liver of vaccination-protected mice in response to early patent infections of Plasmodium chabaudi blood-stage malaria

status: Public on Jun 06 2018

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

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

summary:
The role of the liver for survival of blood-stage malaria is only fragmentary understood.
In the experimental blood-stage malaria Plasmodium chabaudi, protective vaccination induces self-healing and, thus,
survival of otherwise lethal infections. This study investigates the response of the liver, in terms of mRNA and lincRNA expression,
to vaccination-induced self-healing infections and lethal P. chabaudi malaria at early patency on day 4 p.i.,
when parasitized erythrocytes begin to appear in peripheral blood. Using gene expression microarrays,
23 genes were identified to be induced in the liver by >10-fold at p<0.01.
More than one third were genes involved in erythropoiesis, as e.g. Kel, Rhag, Ahsp, Ermap, Slc4a1, Cldn13 Gata1, and Gfi1b.
Another group of >10-fold expressed genes  contain genes involved in natural cytotoxicity,
as those encoding the killer cell lectin-like like receptors Klrb1a, Klrc3, Klrd1, Ncr1 and the granzyme B encoding Gzmb.
In addition, there were identified a series of genes involved in the control of cell cycle and mitosis,
as Ccnb1, Cdc25c, Ckap2l expressed by >10-fold only in vaccination-protected mice, and 22 genes being at least 100% higher expressed
in vaccination-protected mice than the corresponding genes significantly expressed in non-vaccinated mice.
Furthermore, distinct lincRNA species were changed by >3-fold in livers of vaccination-protected mice,
whereas lethal malaria induced different lincRNAs. Our data suggest that protective vaccination accelerates extramedullary erythropoiesis,
generation of liver-resident cytotoxic cells, and regeneration from malaria-induced injuries in the liver at early patency,
which may be critical for final survival of otherwise lethal blood-stage malaria of P. chabaudi.
Mice:
Balb/c mice breeded under specified pathogen-free conditions were delivered from central animal facilities of the University of Düsseldorf.
The experiments were performed only with female mice aged 10-12 weeks.
They were housed in plastic cages, received a standard diet (Woehrlin, Bad Salzuflen, Germany) and water ad libitum.
This study was carried out in strict accordance with the German law on animal protection.
The keeping of mice as well as the experimental protocol of the study were officially approved by the State-controlled Committee on the Ethics of Animal Experiments of the State Nordrhein-Westfalen,
Germany, and were regularly controlled, without being previously announced, by the local authorities.
All efforts were undertaken to minimize suffering of mice.
P. chabaudi malaria infection:
Blood-stage infections of P. chabaudi were kept in outbred mice under sterile conditions by weekly passages of infected blood.
A non-clonal line of P. chabaudi is used in our laboratory since 1982,
which resembles to P. chabaudi chabaudi. Challenge of Balb/c mice with 10^6 P. chabaudi-infected erythrocytes,
Protective vaccination:
As a vaccine, host cell plasma membranes were used, which were isolated in the form of ghosts from P. chabaudi-parasitized erythrocytes
Approximately 10^6 ghosts were suspended in 100 µl Freund’s complete adjuvant (FCA) and subcutaneously injected on week 3 and week 1
before infecting with P. chabaudi-parasitized erythrocytes. Control mice were treated in parallel only FCA.

overall_design: 12 samples were analyzed: Nd0, Nd4, Vd0, Vd4.  3 replicates each.

first: Saleh

last: Al-Quraishy

first: Mohamed

last: Dkhil

first: Abdel

middle: A

last: Abdel-Baki

first: Denis

last: Delic

first: Frank

last: Wunderlich


title: Locus-specific control of the de novo DNA methylation pathway [smRNA-seq]

status: Public on Apr 14 2018

city: San Diego

first: Julie

last: Law

address: 10010 N Torrey Pines Rd, La Jolla

institute: Salk Institute for Biological Studies

summary: Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.

overall_design: 45 smRNA-seq libraries were sequenced

first: Ming

last: Zhou


title: The Quest for Epigenetic regulation underlying unisexual flower development in Cucumis melo

status: Public on May 15 2017

city: Thuwal

first: Prof. Heribert

last: HIRT

address: 4700 KAUST

country: Saudi Arabia

institute: KAUST

summary: Organ-specific landscapes of H3K9ac and H3K27me3 in melon

overall_design: Genome wide H3K27me3 and H3K9ac histone modifications and gene expression dynamics in five melon organs

first: David

last: Latrasse

first: Natalia

last: Rodriguez-Granados

first: Alaguraj

last: Veluchamy

first: Kiruthiga

last: Mariappan

first: Claudia

last: Bevilacqua

first: Nicolas

last: Crapart

first: Céline

last: Camps

first: Vivien

last: Sommard

first: Cecile

last: Raynaud

first: Catherine

last: Dogimont

first: Adnane

last: Boualem

first: Moussa

last: Benhamed

first: Abdelhafid

last: Bendahmane


title: Liver samples of DO mice

status: Public on Sep 26 2017

city: Ithaca

first: Praveen

last: Sethupathy

state: NY

address: Cornell University

institute: Cornell University

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

sample_organism:
Mus musculus
unidentified

overall_design: Refer to individual Series


title: CRISPR/Cas9 genome editing in human neural progenitor cells demonstrates astrocyte pathology in frontotemporal dementia caused by mutant TAU protein

status: Public on Jun 12 2017

first: Marcos

last: Araúzo-Bravo

summary:
Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the MAPT gene encoding TAU. Here, we applied a stem cell model of FTD to examine if FTD astrocytes carry an intrinsic propensity to degeneration and to determine if they can induce non-cell-autonomous effects in neighboring neurons. We utilized CRISPR/Cas9 technology in human iPS cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU, demonstrated proteasome activation and increased oxidative stress and exhibited disease-associated changes in transcriptome profiles. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study expands the utility of CRISPR/Cas9 genome editing to human NPCs to study the pathogenesis of neurodegenerative diseases such as FTD.
For whole genome expression analyses, RNA was isolated from cell lysates using the RNeasy kit (Qiagen) with on-column DNA digestion. 300 ng of total RNA was used as input for cRNA synthesis with the linear amplification protocol (Ambion) comprising the synthesis of T7-linked double-stranded cDNA and in vitro transcription for 12 hr incorporating biotin-labeled nucleotides. Following purification, labelled cRNA samples were hybridized onto HumanHT-12 v4 expression BeadChips (Illumina) for 18 hr following the manufacturer's protocol. The BeadChips were washed as recommended and subsequently stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the associated software. All samples were hybridized as biological replicates. Data were processed by mapping bead intensities to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was achieved by applying the Affymetrix Robust Multiarray Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling. Gene expression normalization was calculated using the quantile method implemented in the lumi package of R-Bioconductor. In-house developed functions in MATLAB were utilized for data post-processing and the generation of graphical representations. Hierarchical clustering analysis of genes and samples was performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method as previously described.

overall_design:
30 samples were analyzed:
Ctrl-Neurons on FTD Astro, GFP-labeled neurons differentiated from C1 neural progenitor cells, which have been co-cultured with FTD1 Astrocytes, 3 replicates;
C1 FTD-2, 3 replicates;
Ctrl-Neurons on Ctrl Astro, GFP-labeled neurons differentiated from C1 neural progenitor cells, which have been co-cultured with Ctrl-2 Astrocytes, 3 replicates;
FTD-1 Astro, Astrocytes differentiated from FTD-1 neural progenitor cells, 3 replicates;
FTD-2 Astro, Astrocytes differentiated from FTD-2 neural progenitor cells, 3 replicates;
Ctrl-2 Astro, Astrocytes differentiated from FTD-1 GC-1 neural progenitor cells, 3 replicates;
Ctrl-1 Astro, Astrocytes differentiated from C1 neural progenitor cells, 3 replicates;
FTD-1, Neural progenitor cells differentiated from iPS cells carrying the N279K MAPT mutation, line 1, 2 replicates;
FTD-2, Neural progenitor cells differentiated from iPS cells carrying the N279K MAPT mutation, line 2, 2 replicates;
C1, Neural progenitor cells differentiated from iPS cells from a healthy control (Ctrl) individual 1, 1 replicate;
FTD-1 GC-1, Control (Ctrl) neural progenitor cells, which have been derived from FTD-1 neural progenitor cells via CRISPR/Cas9-mediated genome editing, line 1, 1 replicate;
FTD-2 GC-1, Control (Ctrl) neural progenitor cells, which have been derived from FTD-2 neural progenitor cells via CRISPR/Cas9-mediated genome editing, line 1, 1 replicate;
FTD-2 GC-2, Control (Ctrl) neural progenitor cells, which have been derived from FTD-1 neural progenitor cells via CRISPR/Cas9-mediated genome editing, line 2, 1 replicate;
FTD-2 GC-3, Control (Ctrl) neural progenitor cells, which have been derived from FTD-1 neural progenitor cells via CRISPR/Cas9-mediated genome editing, line 3, 1 replicate.

first: Anna

last: Hallmann

first: Lampros

last: Mavrommatis

first: Marc

last: Ehrlich

first: Albrecht

last: Rӧpke

first: Johannes

last: Brockhaus

first: Silvia

last: Bolognin

first: Markus

last: Missler

first: Jens

last: Schwamborn

first: Jared

last: Sterneckert

first: Hans

middle: R

last: Schöler

first: Tanja

last: Kuhlmann

first: Holm

last: Zaehres

first: Gunnar

last: Hargus


title: MTF2 recruits Polycomb Repressive Complex 2 by helical shape-selective DNA binding

status: Public on Apr 09 2018

city: Nijmegen

first: Gert

middle: Jan

last: Veenstra

state: Not US or Canada

address: Geert Grooteplein 28

department: Molecular Developmental Biology

institute: Radboud University

summary: Polycomb repression of gene expression is essential for development, with a pivotal role played by trimethylation of histone H3 lysine 27 (H3K27me3) that is deposited by Polycomb Repressive Complex 2 (PRC2). The mechanism by which PRC2 is recruited to target genes has remained largely elusive, in particular in vertebrates. Here we demonstrate that MTF2, one of the three vertebrate homologs of Drosophila Polycomblike, is a DNA-binding, methylation-sensitive PRC2 recruiter in mouse embryonic stem cells. MTF2 directly binds to DNA and is essential for recruitment of PRC2 both in vitro and in vivo. Genome-wide recruitment of the PRC2 catalytic subunit EZH2 is abrogated in MTF2 knock-out cells, resulting in largely reduced H3K27me3 deposition. MTF2 selectively binds regions with a high density of unmethylated CpGs in a context of reduced helix twist, which distinguishes target from non-target CpG islands. These results demonstrate instructive recruitment of PRC2 to genomic targets by MTF2.

overall_design: ChIP-seq for Mtf2, Ezh2, H3K27me3, H3K4me3, Jarid2 and input for reference performed in WT, Mtf2, Jarid2 and Eed mutant mouse embryonic stem cells.

first: Matteo

last: Perino

first: Guido

last: van Mierlo

first: Ino

middle: D

last: Karemaker

first: Siebe

last: van Genesen

first: Michiel

last: Vermeulen

first: Hendrik

last: Marks

first: Simon

last: van Heeringen


title: Global gene expression analysis highlights microgravity sensitive key genes in soleus and EDL of 30 days space flown mice

status: Public on Jan 15 2017

city: Berlin

first: Dieter

last: Blottner

address: Phillipstrasse 12

department: Anatomy

institute: Charité Universitaetsmedizin Berlin

summary:
Microgravity exposure as well as chronic muscle disuse are two of the main causes of physiological adaptive skeletal muscle atrophy in humans and murine animals in physiological condition. The aim of this study was to investigate, at both morphological and global gene expression level, skeletal muscle adaptation to microgravity in mouse soleus and extensor digitorum longus (EDL). Adult male mice C57BL/N6 were flown aboard the BION-M1 biosatellite for 30 days on orbit (BF) or housed in a replicate flight habitat on Earth (BG) as reference flight control.
In this study, we investigated for the first time gene expression adaptation to 30 days of microgravity exposure in mouse soleus and EDL, highlighting potential new targets for improvement of countermeasures able to ameliorate or even prevent microgravity-induced atrophy in future spaceflights.

overall_design: C57BL/N6 mice were randomly divided in 3 groups: Bion Flown (BF), mice flown aboard the Bion M1 biosatellite in microgravity environment for 30 days; Bion Ground (BG), mice housed in the same habitat of flown animals but exposed to earth gravity; and Flight Control (FC), mice housed in a standard animal facility.

last: Gambara

first: Michele

last: Salanova

first: Ute

last: Ungethüm


title: First round of DNA replication is essential for transcriptional reprogramming of somatic nuclei in mouse oocytes

status: Public on Dec 17 2016

first: Marcos

last: Araúzo-Bravo

summary: Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

overall_design:
13 samples were analyzed.
Aph-IVF: Aphidicolin not treated, in vitro fertilization, 1 biological rep
Aph-IVF96: Aphidicolin not treated, in vitro fertilization (96h),  1 biological rep
Aph-IVFBla: Aphidicolin not treated, in vitro fertilization blastocyst,  1 biological rep
Aph+IVF:  Aphidicolin treated, in vitro fertilization, 1 biological rep
Aph+IVF96: Aphidicolin treated, in vitro fertilization (96h),   1 biological rep
Aph+IVFBla: Aphidicolin treated, in vitro fertilization blastocyst,  1 biological rep
Aph-SCNT: Aphidicolin not treated, somatic cell nuclear transfer, 1 biological rep
Aph-SCNT96: Aphidicolin not treated, somatic cell nuclear transfer (96h), 1 biological rep
Aph-SCNTBla:  Aphidicolin not treated, somatic cell nuclear transfer blastocyst, 1 biological rep
Aph+SCNTBla: Aphidicolin treated, somatic cell nuclear transfer blastocyst, 1 biological rep
B6C3F1-Ooc: Aphidicolin not treated, B6C3F1 oocytes, 1 biological rep
B6C3F1-Cum: Aphidicolin not treated, B6C3F1 cumulus cells, 2 biological rep

first: Bingyuan

last: Wang

first: Martin

last: Pfeiffer

first: Caroline

last: Schwarzer

last: Arauzo-Bravo

last: Boiani


title: Dermal myogenic precursors derive from muscle satellite stem cells

status: Public on Feb 21 2017

summary:
The contribution of non-resident, non-satellite myogenic progenitors to postnatal muscle homeostasis and repair is controversial. Precursor cells with the capacity to generate striated muscle fibers in vitro have been isolated from diverse adult tissues, although their physiological role being currently unclear. Since murine dermis-derived precursor cell cultures generate striated muscle when transplanted in vivo, we pursued to identify and characterize the myogenic cell population present in dermis-derived sphere cultures. Lineage tracing experiments for myogenic, perivascular and dermal precursor cell lineages showed a major contribution of Myf5 and Pax7-positive cell progeny to the dermal myogenic precursor cell subset. Tracing, in situ localization and ultrastructural analyses unequivocally demonstrated that Panniculus carnosus muscle-derived satellite stem cells expand in the dermal sphere culture conditions and originate dermis-derived myofibers in vitro. These results highlight the importance of unraveling distinct lineages in sphere cultures to avoid wrong assumptions when determining the developmental potential of adult stem cells.
strain: Crl:CD1(ICR), B6.129S4-Myf5tm3(cre)Sor /J, B6,FVB-Tg(Cspg4-cre)1Akik/J, B195AP-Cre

overall_design:
7 samples were analyzed
Myf5SOR+ Sdc4+, Lineage tracing of mouse Myf5SOR+ [B6.129S4-Myf5tm3(cre)Sor /J] Sdc4+, 1 replicate
Cspg4- Sdc4+, Lineage tracing of mouse Cspg4- [Fullname strain: B6,FVB-Tg(Cspg4-cre)1Akik/J] Sdc4+, 1 replicate
CD1 Sdc4+, Lineage tracing of mouse CD1 [Fullname strain: Crl:CD1(ICR)] Sdc4+, 1 replicate
B195AP+ Sdc4-, Lineage tracing of mouse B195AP+ [Fullname strain: B195AP-Cre]  Sdc4-, 1 replicate
Cspg4- Sdc4-, Lineage tracing of mouse Cspg4- [Fullname strain: B6,FVB-Tg(Cspg4-cre)1Akik/J] Sdc4-, 1 replicate
Cspg4+, Lineage tracing of mouse Cspg4+ [Fullname strain: B6,FVB-Tg(Cspg4-cre)1Akik/J], 1 replicate
CD1 Sdc4-, Lineage tracing of mouse CD1 [Fullname strain: Crl:CD1(ICR)] Sdc4-, 1 replicate
Total RNA was extracted from cells by miRNeasy Micro Kit (50) (217084, Qiagen) and a range between 10 ng and 200 ng of total DNA-free RNA was used as input for labelled cRNA synthesis with the Low Input Quick Amp Labeling Kit (5190-2305, Agilent Technologies), according to manufacturer´s instructions. These cRNA was used on a transcriptome analysis by Agilent SurePrint G3 Mouse GE 8x60K Microarray Kit. Microarray analysis was performed with 8-10 animals per phenotype.

first: Neia

last: Naldaiz-Gastesi

first: Patricia

last: García-Parra

first: María

last: Goicoechea

first: Sonia

last: Alonso-Martín

first: Ana

last: Aiastui

first: Macarena

last: López-Mayorga

first: Paula

last: García-Belda

first: Jaione

last: Lacalle

first: Carlos

last: San José

first: Véronique

last: Le Berre

first: Ander

last: Matheu

first: José

last: García-Verdugo

first: Jaime

last: Carvajal

first: Frédéric

last: Relaix

first: Adolfo

last: López de Munain

last: Izeta


title: DAS graphene-based feeder-free culture system for human induced pluripotent stem cells

status: Public on Feb 09 2017

first: Marcos

last: Araúzo-Bravo

summary:
Graphene has been selected as a candidate for synthetic feeder-free culture substrate guiding human/mouse multipotent stem cell lineage specification, and culturing pluripotent stem cells in a number of studies. However, conventional graphene is not an ideal biomaterial to maintain the pluripotency of human pluripotent stem cells (hPSC) including hESCs/hiPSCs due to its intrinsic hydrophobicity and relatively flat surface topography. Here, we applied morphology-controlled nanocrystalline graphene (NG) coating onto the culture substrates via diffusion-associated synthesis (DAS) process and cultivated hPSCs. It is found that enhanced hydrophilicity and controlled surface roughness of DAS-NG enabled tight focal adhesion of hPSCs onto the DAS-NG coated culture substrate and retained pluripotency for over 2 weeks. It is also found hPSCs grown on DAS-NG shared comparable global gene expression profile with hPSCs grown on mouse embryonic fibroblast (MEF). Importantly, the similarities in cell adhesion gene expression between hPSCs grown on DAS-NG and hPSCs on MEF suggest DAS-NG may provide comparable physical cues with MEF for sustaining pluripotency. Taken together, our findings show a new reliable method for culturing hPSCs in feeder-free condition using DAS-graphene.
Human iPSCs and human ESC H9 were seeded onto DAS-NG coated glass, ITO or QU, CVD-grown graphene coated glass, uncoated glass and mitomycin-C treated CF1. Human pluripotent stem cells seeded on each culture substrate were cultured in human embryonic stem cell medium composed of knockout DMEM (GIBCO-10829) supplemented with 20% knockout serum replacement (GIBCO-10828), 1mM L-glutamine, 1% penicillin/streptomycin (PAA-P11-010), 1% MEM-non essential amino acid (PAA-M11-003), 0.1mM beta-mercaptoethanol, and 5ng/ml human basic fibroblast growth factor.

overall_design:
4 samples were analyzed:
[1] hESC-MEF, H9 human embryonic stem cells (hESCs) grown on mitomycin-C treated MEF, 1 replicate.
[2] hiPSC_MEF, human induced pluripotent stem cells (hiPSCs) grown on mitomycin-C treated MEF, 1 replicate.
[3] hiPSC-DAS/GL, human induced pluripotent stem cells (hiPSCs) grown on diffusion-associated synthesis nanocrystalline graphene (DAS-NG) coated glass, 1 replicate.
[4] hiPSC-w/o DAS, human induced pluripotent stem cells (hiPSCs) grown on uncoated glass, 1 replicate.

first: Donggyu

last: Nam

first: Hyunah

last: Lee

first: Jae

middle: K

last: Choi

first: Myung

last: Park

first: Soon

last: Kwon

first: Jeong

last: Kim


title: N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

status: Public on Nov 25 2016

city: Gent

first: Petra

middle: Van

last: Damme

state: East Flanders

address: Albert Baertsoenkaai 3

department: Department of Biochemistry

country: Belgium

institute: University of Gent

summary: Proteogenomics is an emerging research field yet lacking a standard method of analysis. In this article, we demonstrate the strength of proteogenomic analysis specific for N-terminal data that aims at the discovery of novel translational start sites. In summary, unidentified spectra were matched to a specific N-terminal peptide library encompassing all theoretical protein N-termini encoded in the genome. Gene prediction suggested 81 protein-coding models, of which several alternative proteoforms with unannotated protein starts. Next to the proteomic data, complementary ribosome footprinting data was generated from Arabidopsis thaliana cell cultures. Translation initiation site mapping by the ribosome footprinting data provided orthogonal evidence for 14 novel peptides identified by our proteogenomics pipeline.

overall_design: Ribosome profiling sequencing of Arabidopsis thaliana wild-type ecotype Landsberg erecta cells treated with lactimidomycin or cycloheximide.

first: Patrick

last: Willems

first: Elvis

last: Ndah

first: Veronique

last: Jonckheere

first: Simon

last: Stael

first: Adriaan

last: Sticker

first: Lennart

last: Martens

first: Frank

last: Van Breusegem

first: Kris

last: Gevaert

last: VanDamme


title: Converting Oct6 into a pluripotency inducer by interrogating Oct4 residues

status: Public on Dec 21 2016

last: Araúzo-Bravo

summary: In this study, we set out to identify those molecular features of the POU transcription factor Oct4 that are responsible for inducing pluripotency in somatic cells. Oct4 is known to have a strong preference to cooperate with Sox2 on heterodimeric SoxOct elements predominantly found in enhancers of genes expressed in embryonic stem cells (ESCs). To test whether this partnership is specific to Oct4, we compared its DNA recognition and reprogramming activities to the paralogous transcription factor Oct6, which cannot induce and maintain pluripotency in mouse cells. By analyzing ChIP-Seq data and performing quantitative dimerization assays, we found that in somatic cells, instead of heterodimerzing with Sox-factors, Oct6 more potently homodimerizes on OctOct elements. We identified that a single amino acid is crucial in directing binding to the respective composite DNA element. As a consequence, just changing this one amino acid hampers Oct4 in generating induced pluripotent stem cells (iPSCs). In contrast, the reverse mutation in Oct6 did not augment its reprogramming activity. This was achieved with at least two additional exchanges. In summary, we demonstrate that cell-type specific POU factor function is determined by a limited set of residues that affect DNA and partner factor interactions. Such relatively minor changes lead to a pronounced impact on regulatory function and reprogramming activity.

overall_design:
10 samples were analyzed
MEF, Mouse Embryonic Fibroblast (MEF), 2 replicates
O4SK-iPSC1, Mouse Oct4, Sox2, Klf4 induced Pluripotent Stem Cell (iPSC), line 1, 1 replicate
O4SK-iPSC2, Mouse Oct4, Sox2, Klf4 induced Pluripotent Stem Cell (iPSC), line 2, 1 replicate
OG2-ESC, Mouse OG2 Embryonic Stem Cell (ESC), 2 replicates
O6SKM-iPSC1, Mouse Oct6, Sox2, Klf4, cMyc induced Pluripotent Stem Cell (iPSC), line 1, 1 replicate
O6SKM-iPSC2, Mouse Oct6, Sox2, Klf4, cMyc induced Pluripotent Stem Cell (iPSC), line 2, 1 replicate
O4SKM-iPSC, Mouse Oct4, Sox2, Klf4, cMyc induced Pluripotent Stem Cell (iPSC), 2 replicates

first: Stepan

last: Jerabek

first: Calista

last: Ng

first: Guangming

last: Wu

first: J

first: Kee-Pyo

first: Daniel

last: Esch

first: Vikas

last: Malik

first: Sergiy

last: Velychko

first: Xiaoxiao

last: Yang

first: Vlad

last: Cojocaru

first: Hans

last: Schöler

first: Ralf

last: Jauch


title: Dissecting pluripotency through the different OCT4 and SOX2 assembly configurations

status: Public on Feb 13 2017

first: Marcos

summary: The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). To this end, OCT4 and SOX2 associate and bind to DNA in different configurations depending on the composite DNA element present in their target genes. Here, we have investigated the role of the different OCT4-SOX2 conformational arrangements in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and we have assessed their abilities to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than other OCT4-SOX2 configurations. Overall, the results of this study provide a new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.

overall_design:
Sox2-null ESCs containing a doxycycline-inducible Sox2 transgene, a tet transactivator and a constitutive red-fluorescent protein transgene (DsRed) have been previously described. The different SOX2 mutants were cloned in the hygromycin-resistant PiggyBac plasmid pPBCAG-cHA-IH that is a modification of the previously reported pGG131 plasmid. 500 ng of each plasmid together with 500 ng of pCAG-pBase plasmid were co-transfected into Sox2-null ESCs using Lipofectamine 2000. Next, transfected cells were treated with doxycycline (1 µg/ml) and hygromycin (300 µg/ml). One week later, individual clones were picked and expanded. Finally, RNA was extracted for microarray analysis.

16 samples were analyzed:
S113-4, Sox2-/- ES plus human Sox2 113 mutant clone 4 . in Piggy Bac, plus doxycycline, 2 replicates;
S113-5, Sox2-/- ES plus human Sox2 113 mutant clone 5 overexpressed in Piggy Bac, plus doxycycline, 2 replicates;
SWT-4, Sox2-/- ES plus human Sox2 WT mutant clone 4 overexpressed in Piggy Bac, plus doxycycline, 2 replicates;
SWT-5, Sox2-/- ES plus human Sox2 WT mutant clone 5 overexpressed in Piggy Bac, plus doxycycline, 2 replicates;
S95-1, Sox2-/- ES plus human Sox2 95 mutant clone 1 overexpressed in Piggy Bac, plus doxycycline, 2 replicates;
S95-2, Sox2-/- ES plus human Sox2 95 mutant clone 2 overexpressed in Piggy Bac, plus doxycycline, 2 replicates;
S95-4, Sox2-/- ES plus human Sox2 95 mutant clone 4 overexpressed in Piggy Bac, plus doxycycline, 2 replicates;
TSS, Sox2-/- ES without doxycycline, so mouse Sox2 is expressed, 2 replicates.

first: Natalia

last: Tapia

first: Caitlin

last: MacCarthy

first: Adele

last: Marthaler


title: Development of diagnostic exosomal biomarkers for the detection of invasive non-functional pituitary adenomas (NFPAs)

status: Public on Nov 12 2016

first: Yuan

last: Ren

state: Beijing

address: Dongcheng district, No.1, beishuaifuyuan

department: Neurosurgery

institute: Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College

summary: The incomplete surgical resection of invasive non-functional pituitary adenomas (iNFPAs) carries the increased risk of complications and requires adjuvant radiotherapy and medications. Thus, the molecular mechanisms and markers of invasiveness must be identified to guide the management of NFPA patients. This study explores the proteomic and transcriptomic variations of invasive and non-invasive NFPAs and other types of pituitary adenomas and evaluates the genetic markers in the exosome related to iNFPAs. The exosome from invasive and non-invasive NFPAs, prolactinomas (PRLs), growth hormone–secreting adenomas (GHs), adrenocorticotropic hormone-secreting adenomas (ACTHs), and normal pituitary tissue were analyzed. We confirmed that elevated matrix metalloproteinase-1 (MMP1) expression and its production in the exosome (exo-MMP1) are correlated with the invasive characteristics of NFPA. To investigate the molecular mechanism underlying the role of exo-MMP1 in invasiveness, we analyzed the effects of MMP1 on cell migration, cell growth and tumor angiogenesis. After transfection of MMP1 or a shRNA expression vector into NFPA cells, we obtained the associated exosome and observed that the altered expression and production of MMP1 in the exosome was significantly synchronized with the transduction of NFPA cells. In addition, the enrichment of MMP1 in the exosome promoted cell migration, cell growth and tumor angiogenesis via protease-activated receptor-1 (PAR1) signaling in recipient cells. Thus, these data demonstrate that MMP1 plays an important role in tumor invasion and angiogenesis and show that an exosome-mediated regulatory pathway for MMP1 may represent a target for therapeutic treatment.

overall_design: This study explores the transcriptomic variations of invasive (n=1) and non-invasive NFPAs (n=1) in the exosome.

first: Renzhi

last: Wang


title: Hit and Run Protocol with Growth Factor Enhances the Generation of Mouse Induced Pluripotent Stem Cells

status: Public on Oct 13 2016

summary:
Introducing four transcription factors, Oct4, Sox2, Klf4 and c-Myc can reprogram both human and mouse somatic cells.
first, the oncogenic properties of reprogramming factors such as Klf4 and c-Myc, second, the viral integrations of transcription factors in the genome of somatic cells which may also hinder a clinical setting and finally, the low efficiency of the generation of induced pluripotent stem cells (iPS). Some recent studies overcame the first two problems, the oncogenic properties and genomic integration of the reprogramming factors by either replacing Klf4 and/or c-Myc with small molecules or using different iPS protocols such as integration-free transcription factor inductions as well as introducing proteins. However, the slow reprogramming process and low efficiency of the iPS generation still need to be improved for either clinical application or studying the reprogramming mechanism. Different approaches including adding soluble Wnt3a, and generating iPS cells in hypoxic condition also showed increased efficiency in both 4 factors (Oct4/Sox2/Klf4/c-Myc) or less factors combination without further genetic manipulation. In the current study, we show that basic fibroblast growth factor (bFGF) dramatically increased the efficiency of iPS generation and shortened the reprogramming timing of mouse embryonic fibroblast (MEF) cells in both 4 factors (4F Oct4/Sox2/Klf4/c-Myc) and 3 factors (3F Oct4/Sox2/Klf4) combination without further exogenous genetic manipulation.
RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates.

overall_design: 2 samples were analyzed. Fgf2-:  Mouse embryonic fibroblast culture without Fgf2  and Fgf2+:  Mouse embryonic fibroblast culture without Fgf2

first: Dong

middle: W

last: Han

first: Jin

last: Joo

first: Boris

last: Greber

first: Kinarm

last: Ko

first: Martin

last: Stehling


title: ARTs versus developmental plasticity: blastocyst abnormalities after ICSI and embryo culture do not propagate into the offspring in mice

status: Public on Aug 25 2016

last: Araúzo-Bravo

summary: Following intracytoplasmic sperm injection (ICSI), in vitro culture (IVC) in ART media causes imbalance in the blastocyst's inner cell mass (ICM)with reduction of the primitive endoderm compartment compared to non-IVC blastocystsFollowing embryo transfer (ET), these imbalances do not influence birth rates, and the postnatal effects are inconspicuous. ICSI alters gene expression in mouse embryos. Choice of embryo culture media has been correlated with birth and body growth rates in human studies. No analysis has yet been made combining ICSI and IVC in the mouse. We produced mouse concepti by ICSI followed by IVC and ET so as to mimic,at least in part, the way human embryos are produced and handled in the clinical practice. Fertilized oocytes were cultured in two sets of sequential ART media, compared with an optimized mouse medium and with the mouse oviduct. Embryos were analyzed or implanted to produce newborns. In total, 3706 fertilized mouse oocytes, 1672 blastocysts and 78 newborns were examined during the course of the study. Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and embryo recipients, respectively. B6C3F1 oocytes were fertilized by intracytoplasmic C57Bl/6 sperm injection and were allowed to cleave in HTF/MultiBlast and in ISM1/ISM2 sequential culture protocols as well as in optimized mouse medium (KSOM(aa)) and in vivo in the CD1 mouse oviduct. Cleavage and blastocyst rates were analyzed by Wilcoxon / Kruskal-Wallis test and the blastocysts were split in two subgroups:one subgroup was analyzed for cell lineage allocation and for transcriptome, the other subgroup was transferred to genital tract, allowed to develop to term and characterized for birth rate, litter size, neonatal bodyweight and behavior using ANOVA F-test and t-test. The effects of ART media on blastocyst rates exceed those of ICSI.  Protein and mRNA analysis reveal that IVC has a marked effect on the primitive endoderm lineage of ICSI blastocysts. In contrast to preimplantation, birth rates after ET are indistinguishable not only among ICSI but also in comparison to non-injected controls. At birth, ICSI-IVC-ET concepti are outwardly normal and weigh the same as non-ICSI controls. Confounding effects of subfertility, lifestyle and genetic heterogeneity are reduced to a minimum in the hybrid inbred mouse model compared to human patients. Confounding effects of polyspermy and parthenogenetic activation are prevented by ICSI. There is more to ART than ICSI, IVC and ET, and only a small number of ART media were examined. Preimplantation effects of ART culture media can effectively be modeled in the mouse because the in vitro environment is suboptimal for both mouse and human. However, in the optimal uterine environment of their own species, mouse and human ART blastocysts may be supported and selected for differently, depending on species-specific features such as the kinetics of implantation and the endometrium.

overall_design:
14 samples were analyzed.
HTF/MultiBlast: Mouse multiblastocysts in HTF medium, 3 biological rep
ISM1/ISM2: Mouse blastocysts in ISM1 and ISM2 medium, 3 biological rep
KSOM(aa): Mouse blastocysts in KSOM(aa) medium, 2 biological rep
oviduct: Mouse oviduct, 3 biological rep
micromanipulation control: Mouse micromanipulation control, 3 biological rep

first: Caroline

last: Schwarzer

first: Verena

last: Nordhoff

first: Severine

last: Le Gac

last: Arauzo-Bravo

first: Lisa

last: Trager

first: Lars

last: Lewejohann

first: Michele

last: Boiani


title: Small RNA sequencing on liver samples of DO mice

first: Praveen

last: Sethupathy

summary: Genetic variation, in addition to environmental influences like diet, can govern the expression levels of microRNAs (miRNAs). MiRNAs are commonly found to operate cooperatively in groups to regulate gene expression. To investigate this, we combined small RNA sequencing, clinical phenotypes, and microarray data measuring gene expression from an outbred mouse model, the Diversity Outbred population. In the DO population, each individual has a distinct genome that is a mosaic of 8 inbred founder strains. We used these data to identify co-regulated modules of miRNAs and genes that are influenced by genetics and diet, and identify relationships between the modules and phenotypes in over 200 DO mice.

overall_design: Investigation of differences in microRNA expression in DO mice fed either a high-fat, high-cholesterol diet with added cholic acid (HFCA), or a calorie-match high protein (HP) diet used as a control, for 18 weeks. One of each sibling pair was placed on either diet

first: Alisha

last: Coffey

first: Tangi

last: Smallwood

first: Jody

last: Albright

first: Matt

last: Kanke

first: Kunjie

last: Hua

first: Daniel

last: Pomp

first: Brian

last: Bennett


title: Expression data in an Arabidopsis cytokinin (CK)-signaling mutant, ahp2,3,5 and arr1,10,12, and its wild type plant (WT, Col-0) under high salinity (200 mM NaCl) and control (0 mM NaCl) conditions.

status: Public on Dec 31 2017

city: Yokohama

first: Rie

last: Nishiyama

address: Tsurumi, Suehiro-cho 1-7-22

department: CSRS

country: Japan

institute: Riken

summary: To understand the role of CK-signaling components, AHPs (His-containing phosphotransfer proteins) and type-B ARRs (response regulators) in salt stress response, we have employed transcriptional profiling of ahp2,3,5 and arr1,10,12, and it's wild type plant (WT), Col-0 under high salinity (200 mM NaCl) and control (0 mM NaCl) conditions. Agilent’s Whole Arabidopsis Gene Expression Microarray (G2519F-021169, V4, 4x44K) was used.

overall_design: 10-day-old ahp2,3,5, arr1,10,12, and Col-0 plants grown on MS media to were transferred to 0.5 x MS supplemented with 200 mM NaCl (salt stress) or 0 mM NaCl (control), collected just after 24 hours. Total RNA was prepared and used for the microarray hybridization. Three replicative hybridization experiments were carried out for each independent biological sample.

first: Yasuko

last: Watanabe

first: Maho

last: Tanaka

first: Motoaki

last: Seki

first: Lam-Son

middle: P

last: Tran


title: Global transcript structure resolution of high gene density genomes through multi-platform data integration: deepCAGE

status: Public on Jul 08 2016

city: New Orleans

first: Erik

last: Flemington

state: LA

address: 1430 Tulane Ave., SL79

institute: Tulane Health Sciences Center

summary: deepCAGE was used in conjunction with Pacific Biosciences Iso-Seq and Illumina RNA-Seq to globally resolve transcript structures in replicating Epstein-Barr virus.

overall_design: deepCAGE of replicating Epstein-Barr virus in Akata cells to identify transcript 5' ends

first: Tina

last: O'Grady

first: Xia

last: Wang

first: Kerstin

last: Höner zu Bentrup

first: Melody

last: Baddoo

first: Monica

last: Concha


title: Global transcript structure resolution of high gene density genomes through multi-platform data integration: Illumina RNA-Seq

summary: Strand-specific Illumina RNA-Seq was used in conjunction with Pacific Biosciences Iso-Seq and deepCAGE to globally resolve transcript structures in replicating Epstein-Barr virus.

overall_design: Illumina strand-specific RNA-seq of BCR-activated Akata cells at 9 time points, and GapmeR knockdown of BZLT12-22 transcripts


title: An in vitro human liver model by iPSC-derived parenchymal and non-parenchymal cells

status: Public on Jul 27 2017

city: Bunkyo-ku

first: Taketomo

last: Kido

state: Tokyo

address: 1-1-1 Yayoi

department: Institute for Quantitative Biosciences

institute: The University of Tokyo

summary: We have established culture systems to generate liver progenitor cells (LPCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) from hiPSCs. Then, we established co-culture system of hiPSC-derived liver cells and performed RNA-seq of hiPSC-derived liver model.

overall_design: Liver progenitor cells (LPCs), Liver sinusoidal endothelial cells (LSECs, and hepatic stellate cells (HSCs) were generated from human iPS cells. Expression profile of hiPSC-derived liver model (co-culture system of LPCs and NPCs, N=2) was compared with hiPSC-derived LPCs (N=2) and primary human hepatocytes (N=2) by RNA-seq.

first: Yuta

last: Koui


title: The germline-specific factor OEF-1 facilitates coordinated progression through germ cell development in C. elegans

status: Public on Nov 22 2017

city: New Haven

first: Valerie

last: Reinke

state: CT

address: 333 Cedar St

department: Genetics

institute: Yale University School of Medicine

summary: How germ cells faithfully pass genetic material to the next generation despite undergoing several developmental transitions during maturation is poorly understood. Here we identify a novel factor in C. elegans, OEF-1, that is highly germline-specific, with early expression in the embryonic germ lineage and differential expression in larvae and adults depending on the gametogenesis program. OEF-1 is nuclear and associates with autosomal germline-expressed genes. Loss of OEF-1 triggers accelerated germ cell progression, leading to defects at multiple points during germline development. Thus OEF-1 may act to coordinate the timely progression of germ cells through proliferation, meiotic entry, and gametogenesis.

overall_design: This data submission includes a single ChIP-seq sample for one factor called OEF-1 in C. elegans. These samples were done in replicate, with input, yielding three sequence files per sample (two replicates, and one input fasta file). In addition, we are submitting two wig files for the dataset, consisting of one normalized wig file for the combined duplicate IP and one normalized wig file for the input sequence.

first: Catherine

middle: E

last: McManus


title: A TAD boundary at the HoxD locus segregates opposing limb regulatory landscapes and their target genes [ChIP-Seq]

status: Public on Dec 22 2017

first: Eddie

last: Rodríguez-Carballo

address: 4, Boulevard d'Yvoy

institute: Université de Genève

summary: The mammalian HoxD cluster is positioned at the boundary between two topologically associating domains (TADs), each of them matching a distinct, enhancer-rich regulatory landscape. During limb development, the telomeric TAD controls the early phase of Hoxd gene transcription in future forearm cells, whereas the centromeric TAD subsequently regulates transcription of more posterior Hoxd genes in presumptive digit cells. The TAD boundary is essential as it prevents the terminal Hoxd13 gene to respond to potent forearm enhancers, thereby allowing the formation of proper proximal limb structures. Here we apply chromosome conformation capture onto embryonic proximal and distal limb bud cells micro-dissected from a set of nested deletions involving part- or all- of this boundary region to try to understand the nature and function of this CTCF- and cohesin-rich DNA region. We document a progressive release of the boundary effect, allowing for inter-TAD contacts to be established, which were favoured by the functional status of the newly accessed enhancers. However, the boundary was highly resilient and only a 400kb large deletion including the whole gene cluster was eventually able to merge the two neighbouring TADs into a single structure. We propose that the whole HoxD cluster is a dynamic transcriptional boundary, showing slight variations depending on both the transcriptional status and the ontogenetic context

overall_design: ChIP analysis of CTCF, SMC1, RAD21 and H3K27ac E12.5 limb tissues from Wt or serial HoxD-deleted alleles

first: E

last: Rodriguez-Carballo

first: L

last: Lopez-Delisle

last: Beccari

first: D

last: Duboule


title: H3K27me3 ChIP-seq in rat peripheral nerve

status: Public on Aug 01 2016

city: Madison

first: John

last: Svaren

address: 1500 Highland Ave.

institute: University of Wisconsin

summary: ChIP-seq of H3K27me3 in rat peripheral nerve was used to identify sites of polycomb repression associated with genes in Schwann cells, which constitute the majority of cells in peripheral nerve.

overall_design: H3K27me3 ChIP samples were prepared from rat sciatic nerve and then sequenced. Inputs for these ChIP samples have previously been submitted as samples GSM2858934 and GSM2858933.

first: Ki

middle: H

last: Ma


title: Altered expression of long noncoding RNAs in LNCaP castration resistant prostate cancer cell lines

status: Public on Aug 18 2017

city: Guangzhou

first: Xu

last: Chen

state: Guangdong

address: 107. W. Yanjiang Road

department: Department of Urology

institute: Sun Yat-sen Memorial Hospital

summary: Castration-resistant prostate cancer (CRPC) that arise after the failure of androgen deprivation therapy is a leading cause of deaths in prostate cancer patients.However, its underlying mechanism is not fully understood. Long noncoding RNAs (lncRNAs) act as crucial regulators in a lot of human cancers, yet their potential roles and molecular mechanisms in CRPC are poorly understood.The goal of this study is to identify the differentially expressed lncRNAs in LNCaP cells and its two castration resistant sublines. Our study reveals that deregulation of lncRNAs is involved in the initiation and progression of CRPC.

overall_design: We constructed two LNCaP castration resistant sublines.On one hand, we generated LNCaP-Bic by continuously exposed to bicalutmide for more than 12 months.On the other hand, LNCaP-AI was established by sustained passaging in cultural medium supplied with androgen deprived serum. The RNA of the cells was extracted and hybridized on Affymetrix microarrays. We indentified the differentially expressed lncRNAs in LNCaP-Bic and LNCaP-AI cells, as compared with LNCaP. We futher validated the data of microarray by RT-qPCR.

first: Peng

last: Gu

first: Tianxin

last: Lin

first: Jian

last: Huang


title: Pericyte Characteristics and an Optimal Factor Combination Enable Generating Bona-Fide iOPCs with the High Efficiency

status: Public on May 21 2017

first: Marcos

last: Araúzo-Bravo

summary: The possibility to generate induced oligodendrocyte progenitor cells (iOPCs) from fibroblasts may enable in vitro neurological disease modeling and autologous remyelinating therapies.

overall_design:
10 samples were analyzed
PC  Pericyte 2 replicates
OPC  Primary Oligodendrocyte 2 replicates
PC2FiOPCp5  Induced oligodendrocyte progenitor cells from pericyte after 5 passages 2 replicates
PC2FiOPCp15  Induced oligodendrocyte progenitor cells from pericyte after 15 passages 2 replicates
PC2FiOPCp25  Induced oligodendrocyte progenitor cells from pericyte after 25 passages 2 replicates

first: Kee-Pyo

last: Kim

first: Juyong

last: Yoon

first: Jong-Wan

first: Jin

middle: Y

last: Hong

first: Gunnar

last: Hargus

first: Anna

last: Preisner

first: Karin

last: Hagemeier

first: Martin

last: Stehling

first: Holm

last: Zaehres

first: Ji-hoon

last: Yang

first: Hyun-Woo

last: Jeong

first: Tae-Hwan

last: Kwak

first: Dong

last: Han

first: Ralf

last: Adams

first: Tanja

last: Kuhlmann

first: Jung-Keun

last: Hyun

first: Hans

last: Schöler


title: Microarray measuring gene expression in livers of DO mice

status: Public on Sep 26 2017

first: Praveen

last: Sethupathy

overall_design: DO mice were fed either a high-fat, high-cholesterol diet with added cholic acid (HFCA), or a calorie-match high protein (HP) diet used as a control, for 18 weeks. One of each sibling pair was placed on either diet.

first: Alisha

last: Coffey

first: Tangi

last: Smallwood

first: Jody

last: Albright

first: Matt

last: Kanke

first: Kunjie

last: Hua

first: Daniel

last: Pomp

first: Brian

last: Bennett


title: Global transcript structure resolution of high gene density genomes through multi-platform data integration: Iso-Seq

first: Erik

last: Flemington

summary: Pacific Biosciences Iso-Seq was used in conjunction with Illumina RNA-Seq and deepCAGE to globally resolve transcript structures in replicating Epstein-Barr virus.

overall_design: Iso-Seq of induced Akata cells

first: Tina

last: O'Grady

first: Xia

last: Wang

first: Kerstin

last: Höner zu Bentrup

first: Melody

last: Baddoo

first: Monica

last: Concha


title: GAA deficiency in Pompe disease is alleviated by exon inclusion in iPS cell-derived skeletal muscle cells

status: Public on Apr 11 2017

first: Marcos

last: Araúzo-Bravo

summary: While there are many human skeletal muscle disorders, very few therapies have been developed. It has not been possible to generate large amounts of purified skeletal muscle cells from pluripotent stem cells, and to test therapies quantitatively. We therefore devised conditions for generating and expanding purified human myogenic progenitors from induced pluripotent stem (iPS) cells. The progenitors retained the capacity to differentiate into multinucleated myotubes and showed a normal karyotype throughout the expansion phase. We applied this method to Pompe disease, a metabolic myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). In a screen, we identified sequences that suppressed aberrant GAA exon 2 splicing caused by the frequent c.-32-13T>G (IVS1) GAA variant. Antisense oligonucleotides (AONs) that blocked these sequences promoted exon 2 inclusion in patient-derived myotubes. As this raised GAA enzymatic activity above the disease threshold, AON-mediated splicing correction may provide a treatment option for Pompe disease.

overall_design:
RNA samples to be analyzed by microarrays were prepared using RNeasy columns with on-column DNA digestion (Qiagen). 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto HumanHT-12 v4 expression BeadChips (Illumina) following the manufacturer's instructions. After recommended washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data postprocessing and graphics was performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method.

6 samples were analyzed:
F134, Human fibroblast line F134, 1 replicate
237iPS, Human induced reprogrammed cells (iPSCs) from patient 1, 1 replicate
75iPS, Human induced reprogrammed cells (iPSCs) from patient 2, 1 replicate
1981iPS, Human induced reprogrammed cells (iPSCs) from patient 1, 1 replicate
H1, Human Embryonic Stem Cell (ESC) H1, 1 replicate
H9, Human Embryonic Stem Cell (ESC) H9, 1 replicate

last: van der Wal

first: Atze

last: Bergsma

first: Joon

last: Pijnenburg

first: Tom

last: van Gestel

first: Holm

last: Zaehres

first: Ans

middle: T

last: van der Ploeg

first: W

last: Pijnappel


title: Different reprogramming methods generate undistinguishable pluripotent states

status: Public on Dec 17 2015

summary:
Pluripotency can be induced from different cell types using different methods. Several studies have reported that iPSC retain an epigenetic memory from the tissue from which they were derived. However, the influence of the method used for reprogramming in the final pluripotent state has not been investigated yet. Therefore, we have taken advantage of germline stem cells (GSCs) that can be converted into germline pluripotent stem cells (gPSCs) using specific culture conditions or into induced pluripotent stem cells (iPSCs) after the forced expression of four transcription factors. Here, we compare the gene expression profile, imprinting methylation pattern and differentiation potential between GSC-derived iPSCs and gPSCs. Our analysis demonstrates that both reprogramming methods induce equivalent levels of pluripotency and that a donor GSC transcriptional memory is retained only at early steps after reprogramming. In summary, our results show that different reprogramming methods generate undistinguishable pluripotent states and exclude a method-dependent epigenetic memory.

Cell Culture. All the cell lines used in this study were derived from OG2 mice. Unless otherwise noted, all cell culture reagents were purchased from Gibco or PAA. Neonatal GSCs derived from 10 days postpartum (d.p.p) murine testis and adult GSCs derived from 35 d.p.p murine testis were derived as previously described and cultured on irradiated C3H feeder cells in a medium composed of StemPro-34 SFM (including supplement), N2 supplement (1:100), 1 % heat-inactivated fetal calf serum (FCS), 5 mg ml-1 bovine serum albumin (BSA) fraction V, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L‑glutamine, 0.1 mM non-essential amino acids (NEAA), 0.1 mM β‑mercaptoethanol, MEM vitamins (1:100), 6 mg ml-1 D‑(+)‑glucose (Sigma), 30 mg ml-1 pyruvic acid, 0.1 % DL‑lactic acid (Sigma), 60 ng ml-1 progesterone (Sigma), 30 ng ml-1 β‑estradiol (Sigma), 15 ng ml-1 mouse EGF (Peprotech), 10 ng ml-1 human bFGF (Peprotech), 10 ng ml-1 human GDNF (Peprotech), and 1000 U ml-1 mouse LIF (in‑house preparation). For maintenance culture, GSCs were passaged at a 1:2‑ratio once a week after digestion with 0.05 % trypsin/EDTA. In addition, ESCs, iPSC and gPSC were cultured on irradiated C3H feeders in the following medium: Knockout DMEM, 20% Knockout Serum Replacement, penicillin, streptomycin, L‑glutamine, NEAA, β‑mercaptoethanol, and LIF.
Generation of pluripotent cell lines. iPSC were generated using tetracycline-inducible lentiviruses. For the lentiviral production, 293T cells were plated at 50 % confluency in 10-cm dishes one day prior to transfection. The next day, the lentiviral vector (3 µg) together with the packaging plasmids psPAX2 (2 µg) and pMD2.G (1 µg) (Addgene plasmids #12260 and #12259, respectively) were transfected using FuGENE 6 reagent (Roche) according to the manufacturer’s protocol. After 48 h, viral-containing supernatants were filtered (0.45 µm), concentrated around 30-fold by centrifugation (26,000 × g, 2 h, 4 °C) and stored at -80 °C. For the generation of iPSCs, a single-cell suspension of 50,000 GSCs was infected overnight with five tetracycline-inducible lentiviruses (5–20 µl concentrated supernatant each) encoding for Oct4, Sox2, Klf4, c-Myc, and the reverse tetracycline-transactivator M2-rtTA (Addgene plasmids #20323, #20326, #20322, #20324, and #20342) in the presence of 4 µg ml-1 polybrene (Sigma). One week after the infection, the expression of the viral transgenes was induced by adding 4 µg ml-1 doxycycline (Sigma) to the medium. MEFs-derived iPSCs were generated using the same protocol. gPSC were generated following a previous reported protocol. Briefly, GSCs were seeded at a density of approx. 1000 cells cm-2 on feeder cells and were not splitted until gPSC colonies were observed. Both iPSC and gPSC colonies were identified by Oct4-GFP expression, manually picked and expanded under ESC conditions. In vitro differentiation of pluripotent cells. Embryoid body (EB) formation was induced in suspension culture adopting the hanging drop method (600 cells in 20 µl) for 5 days in ESC medium without LIF. EBs were seeded on gelatine-coated plates and further cultured for 14 days in the same medium. Chimera formation. Aggregations of putative pluripotent cells with denuded 8-cell stage embryos were performed as previously reported. After overnight incubation at 37 °C and 5 % CO2, 11–14 aggregates were transferred into the uterine horns of 2.5 d.p.c pseudo-pregnant surrogate mothers (mated with vasectomized males). The chimeric pups were obtained and dissected at 14.5 d.p.c. Microarray gene expression analysis. Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 hours of in-vitro transcription incorporating biotin-labelled nucleotides. Hybridizations onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturer’s recommendations. Two replicates were hybridized from each sample. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

overall_design:
Samples were hybridised as biological replicates. 26 samples were analyzed.

iPSC[GSCneonatal]#1: iPSC day 10 from GSC, clone 1, 2 biological rep
iPSC[GSCneonatal]#2: iPSC day 10 from GSC, clone 2, 2 biological rep
iPSC[GSCadult]#1:    iPSC day 40 from GSC, clone 1, 2 biological rep
iPSC[GSCadult]#2:    iPSC day 40 from GSC, clone 2, 2 biological rep
gPSC[GSCneonatal]#1: gPSC day 10 from GSC, clone 1, 2 biological rep
gPSC[GSCneonatal]#2: gPSC day 10 from GSC, clone 2, 2 biological rep
gPSC[GSCadult]#1:    gPSC day 40 from GSC, clone 1, 2 biological rep
gPSC[GSCadult]#2:    gPSC day 40 from GSC, clone 2, 2 biological rep
ESC:                 OG2 ESCs, 2 biological rep
iPSC[MEF]:           iPSC (Tet-4F) from OG2 MEFs, 2 biological rep
GSC[neonatal]:       GSC day 10, 2 biological rep
GSC[adult]:          GSC day 40, 2 biological rep
MEF:                 Mouse embryonic fibroblasts, 2 biological rep

first: Ulf

last: Tiemann

first: Guangming

last: Wu

first: Adele

middle: G

last: Marthaler

first: Natalia

last: Tapia

first: Hans

last: Schöler


title: A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

status: Public on May 19 2016

first: Nayuta

last: Yakushiji-Kaminatsui

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

institute: RIKEN Center for Integrative Medical Sciences

summary: During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and, subsequently, into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model whereby the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition.

overall_design: ChIP-seq analysis of H3K27ac and  H3K27me3 in the proximal and distal forelimbs at mouse E12.5

first: N

last: Lonfat

first: D

last: Duboule


title: Identification of early neurodegenerative changes in an induced pluripotent stem cell model of frontotemporal dementia linked to mutant tau protein

status: Public on Feb 10 2017

summary: Frontotemporal dementia (FTD) is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein tau. Due to limited availability of neural cells from patients` brains, however, the underlying mechanisms of neurodegeneration in FTD are still only poorly understood. Here, we derived induced pluripotent stem (iPS) cells from individuals with FTD-associated MAPT mutations and differentiated them into neurons for mechanistic studies. Patient-derived neurons demonstrated pronounced tau pathology with increased fragmentation and AT8-immunoreactivity, disturbed neurite extension and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD-neurons showed activation of the unfolded protein response and presented with distinct, disease-associated gene expression profiles in a whole-genome transcriptome analysis. These findings indicate distinct, early neurodegenerative changes in FTD caused by mutant tau and highlight the unique opportunity to use patient-specific iPS cells to identify potential targets for drug screening purposes and therapeutic intervention. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer’s instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The bead intensities were mapped to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model (Irizarry et al., 2003). Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in MATLAB. Hierarchical clusters of genes and samples were performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method.

overall_design:
28 samples were analyzed
Fib, Human fibroblast F134, 2 replicates
FTDP17 1-I iPSC, Human induced Pluripotent Stem Cell (iPSC) from Frontotemporal Dementia and Parkinsonisom linked to chromosome 17 (FTDP17) patient 1 clone I, 1 replicate
FTDP17 2-I iPSC, Human induced Pluripotent Stem Cell (iPSC) from Frontotemporal Dementia and Parkinsonisom linked to chromosome 17 (FTDP17) patient 2 clone I, 1 replicate
Ctrl 1-I iPSC, Human induced Pluripotent Stem Cell (iPSC) from healthy control 1 clone I, 1 replicate
H1 ESC, H1 human Embryonic Stem Cells (ESCs), 3 replicates
H9 ESC, H9 human Embryonic Stem Cells (ESCs), 3 replicates
Ctrl-2 Dif, Neurons differentiated from human induced Pluripotent Stem Cell (iPSC) from healthy control 2, 3 replicates
Ctrl-1 Dif, Neurons differentiated from human induced Pluripotent Stem Cell (iPSC) from healthy control 1, 3 replicates
FTDP17-1 Dif, Neurons differentiated from human induced Pluripotent Stem Cell (iPSC) from Frontotemporal Dementia and Parkinsonisom linked to chromosome 17 (FTDP17) patient 1, 3 replicates
FTDP17-2 Dif, Neurons differentiated from human induced Pluripotent Stem Cell (iPSC) from Frontotemporal Dementia and Parkinsonisom linked to chromosome 17 (FTDP17) patient 2, 3 replicates
FTDP17 2-II iPSC, Human induced Pluripotent Stem Cell (iPSC) from Frontotemporal Dementia and Parkinsonisom linked to chromosome 17 (FTDP17) patient 2 clone II, 1 replicate
FTDP17 1-II iPSC, Human induced Pluripotent Stem Cell (iPSC) from Frontotemporal Dementia and Parkinsonisom linked to chromosome 17 (FTDP17) patient 1 clone II, 1 replicate
Ctrl 1-II iPSC, Human induced Pluripotent Stem Cell (iPSC) from healthy control 1 clone II, 1 replicate
Ctrl 2-I iPSC, Human induced Pluripotent Stem Cell (iPSC) from healthy control 2 clone I, 1 replicate
Ctrl 3-I iPSC, Human induced Pluripotent Stem Cell (iPSC) from healthy control 2 clone I, 1 replicate

first: Marc

last: Ehrlich

first: Anna-Lena

last: Hallmann

first: Peter

last: Reinhardt

first: Sabrina

last: Korr

first: Albert

last: Röpke

first: Olympia

last: Psataki

first: Petra

last: Ehlig

first: Sven

last: Meuth

first: Bernardino

last: Ghetti

first: Jared

last: Sterneckert

first: Tanja

last: Kuhlmann

first: Gunnar

last: Hargus


title: Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis

status: Public on Feb 22 2016

first: Marcos

last: Araúzo-Bravo

summary: Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine, but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert profibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

overall_design:
Whole Mouse Genome Oligo Microarray v2 (4x44K) (Agilent Technologies) was used to characterize global gene expression profiles of iHeps compared to myofibroblasts and primary mouse hepatocytes. All microarrays were performed at the Research Core Unit Transcriptomics of the Hanover Medical School. Briefly, total RNA was used to prepare the aminoallyl-UTPmodified (aaUTP) cRNAs (Amino Allyl MessageAmp™ II Kit; #AM1753; Life Technologies) as directed by the company. The aaUTP-cRNAs were labelled with Alexa Fluor 555 Reactive Dye (#A32756; LifeTechnologies). Prior to the reverse transcription reaction, 1μl of a 1:5000 dilution of Agilent’s One-Color spike-in Kit stock solution (#5188-5282, Agilent Technologies) was added to 100ng of total RNA of each analyzed sample. The cRNA fragmentation, hybridization, and washing steps were carried out according to Agilent’s One-Color Microarray-Based Gene Expression Analysis Protocol V5.7 except that 500ng of each labelled cRNA sample were used for hybridization. Slides were scanned on the Agilent Micro Array Scanner G2565 CA (pixel resolution 5 μm, bit depth 20). Data extraction was performed with the Feature Extraction Software V10.7.3.1.

12 samples were analyzed:
Pr-mHSC: Primary myofibroblasts derived from hepatic stellate cells (HSCs), 2 replicates;
In-vivo iHep: In-vivo myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates;
In-vivo eHep: In-vivo endogenous hepatocytes (eHep), 3 replicates;
In-vitro iHep: In-vitro myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates;
PH24h: Primary hepatocytes (PH) cultured for 24 hours, 1 replicate.

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