title: The life history of retrocopies illuminates the evolution of new mammalian genes

first: Francesco Nicola

last: Carelli

address: Henry Wellcome Building of Cancer and Developmental Biology,  Tennis Court Road

institute: University of Cambridge

summary: New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as useful models for the study of new gene origination. Here we combine extensive mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, the majority evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers or bivalent regulatory elements, as well as from CpG islands not associated to other genes. Altogether, these mechanisms facilitated the birth of up to 280 retrogenes in each therian species. Furthermore, the regulatory evolution of the originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated the cooption of distant promoters and in many cases allowed the expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute their parents during the process of male (meiotic) X inactivation, while others even rendered parental functions completely superfluous, allowing for parental gene loss. Overall, our reconstruction of the complete “life history” of mammalian retrogenes highlights the usefulness of retroposition as a general model for understanding new gene birth and functional evolution.

overall_design: Assembly and expression of vertebrate retrogene transcripts

first: Francesco

middle: N

first: Takashi

last: Hayakawa

first: Yasuhiro

last: Go

first: Hiroo

last: Imai

first: Maria

last: Warnefors

first: Henrik

last: Kaessmann


title: NSD3-short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler (ChIP-Seq)

status: Public on Dec 17 2015

city: Cold Spring Harbor

first: Christopher

middle: R.

last: Vakoc

state: New York

address: One Bungtown Road

country: USA

institute: Cold Spring Harbor Lab

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by using a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome, and each is released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.

overall_design: ChIP-Seq for regulatory factors of BRD4, NSD3, CHD8 and histone modification H3K36me2 in MLL-AF9 transformed acute myeloid leukemia cells (RN2)

first: Chen

last: Shen

middle: R


title: TELP, a sensitive and versatile library construction method for next-generation sequencing

status: Public on Dec 15 2015

city: Beijing

first: Wei

last: Xie

address: zhongguancun beidajie

country: China

institute: Tsinghua University

type: Expression profiling by high throughput sequencing

summary: Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.

overall_design: Pre-adipocytes and mature adipocytes were collected. Their chromatin and RNA were subjected to ChIP and mRNA extraction.  Sequencing libraries from ChIP DNA or mRNA were generated following either standard protocols or TELP method. The quality and features of TELP libraries were proved and demonstrated in comparison with standard libraries or other published data.

first: Jingyi

last: Wu

first: Xu

last: Peng

first: Feng

last: Xu


title: Trophoblast stem cells (TSC) global transcriptome in stemness and differentiation conditions after treatment with PF1-3

status: Public on Dec 16 2015

city: Freiburg

middle: Josefina

last: Castex

address: BreisacherStrasse 66

country: Germany

institute: Freiburg Uniklinik

summary: Here we report a potent, acetyl-lysine competitive and cell active inhibitor, PFI-3, that tightly binds to the bromodomain present in BRG1/BRM and the fifth bromodomain in Polybromo (BAF180). The high specificity of PFI-3 was achieved based on a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in bromodomain inhibitor complexes. Embryonic stem cells exposure to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was dramatically enhanced.

overall_design: TSC were treated with PF1-3 or control compound in stemness or differentiation conditions, 2 replicates were used for treatment in stemness and differentiation conditions together with 2 control replicates in stemness and one during differentiation; DFKZ genomics and proteomics

first: Thomas

last: Günther

first: Josefina


title: HITS-CLIP of cytoplasmic SLBP RNP

city: Hanover

first: Michael

last: Whitfield

state: NH

address: Geisel School of Medicine; HB 7400

institute: Geisel School of Medicine at Dartmouth

summary: UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP

overall_design: Polyclonal α-SLBP antibody was used to immunoprecipitate UV-crosslinked SLBP RNP from cytoplasmic HeLa lysates. Mock immunoprecipitation were also performed and serve as a negative control.

first: Lionel

last: Brooks III

middle: L


title: A multi-protein occupancy map of the mRNP on the 3' end of histone mRNAs

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

overall_design: Refer to individual Series


title: Combinatorial Control of RBP-jk and EBF1 Chromosome Occupancy induced by viral co-activator EBNA2

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute


title: Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep

city: Columbia

last: Spencer

state: MO

address: 920 East Campus Drive

department: Animal Sciences

institute: University of Missouri

summary: The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs with the potential to mediate conceptus-maternal communication during early pregnancy. In Study One, EVs were purified from uterine luminal fluid (ULF) of day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study Two, day 14 conceptuses were cultured ex vivo for 24 hours and found to release EVs into the culture medium. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. No evidence of EV escape from the uterine lumen was observed by analysis of the ovary and other maternal tissues. Proteomics analysis of the day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes including proteases, protease inhibitors, chaperones and chaperonins. RNA-sequencing of day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top expressed genes were overrepresented in ribosomal functions and components. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication during the establishment of pregnancy.

overall_design: Transcriptional profiles from day 14 conceptus extracellular vesicles isolated from 24 hour conceptus-conditioned culture media (n=3) were generated by sequencing on the Illumina HiSeq 2500 platform.

first: Gregory

middle: W

last: Burns

first: Kelsey

middle: E

last: Brooks


title: A single 3D chromatin compartment in amphioxus Hox cluster suggests a stepwise evolutionary origin for vertebrate Hox bimodal regulatory topologies

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

country: Spain

institute: CABD/CSIC

summary: Comparison among vertebrates and the cephalochordate amphioxus Hox clusters architectures

sample_organism:
Branchiostoma lanceolatum
Danio rerio

platform_organism:
Branchiostoma lanceolatum
Danio rerio

overall_design: 4C-seq experiments from promoters spanning the amphioxus Hox cluster and the zebrafish hoxd cluster and surrounnding areas,

first: Rafael

middle: D

last: Acemel

first: Ibai

last: Irastorza

first: Ferdinand

last: Marlétaz

first: Carlos

last: Gómez-Marín

first: Elisa

last: de la Calle-Mustienes

first: Sophie

last: Bertrand

first: Sergio

middle: G

last: Diaz

first: Peter

last: Holland

first: Damien

middle: P

last: Devos

first: Ignacio

last: Maeso

first: Hector

last: Escrivá

first: José

last: Gómez-Skarmeta


title: Spo11-oligo mapping in S. cerevisiae Ctf19/CCAN kinetochore sub-complex mutant mcm21

city: New York

first: Isabel

last: Lam

address: 1275 York Avenue

department: Molecular Biology

institute: Memorial Sloan Kettering Cancer Center

summary: During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes.

overall_design: Two samples total: two biological replicate Spo11-oligo maps of S. cerevisiae SK1 mcm21 null mutant

first: Nadine

last: Vincenten

first: Lisa-Marie

last: Kuhl

first: Ashwini

last: Oke

first: Alastair

last: Kerr

first: Andreas

last: Hochwagen

first: Jennifer

last: Fung

first: Scott

last: Keeney

first: Gerben

last: Vader

first: Adele

last: Marston


title: CRISPR-Cas9 combinatorial KO of epigenetic regulators in human ovarian cancer cells

city: Boston

first: Gabriela

last: Pregernig

state: Massachusetts

address: 1325 Boylston Street, Suite 500

institute: Decibel Therapeutics

summary: We sequenced polyA mRNA from OVCAR8-ADR-Cas9 cells in which one or two of 3 epigenetic regulators (BRD4, KDM4C, KDM6B) had been knocked out to examine how global gene expression was affected and evaluate potential synergistic effects at a molecular level.

overall_design: Gene expression data (RNA-Seq) in OVCAR8-ADR-Cas9 cells infected with control vector or vectors expressing gRNAs targeting one of 4 epigenetic regulators (BRD4, KDM4C, KDM6B) with biological replicates.

first: Alan

middle: S

last: Wong


title: Competition between DNA methylation and transcription factors determines binding of NRF1

city: Illkirch

first: Anais

middle: Flore

last: Bardet

address: 1 rue Laurent Fries

country: France

institute: IGBMC

type: Methylation profiling by high throughput sequencing

summary: Eukaryotic transcription factors (TFs) are key determinants of gene activity, yet they bind only a fraction of their corresponding DNA sequence motifs in any given cell type. Chromatin has the potential to restrict accessibility of binding sites; however, in which context chromatin states are instructive for TF binding remains mainly unknown. To explore the contribution of DNA methylation to constrained TF binding, we mapped DNase-I-hypersensitive sites in murine stem cells in the presence and absence of DNA methylation. Methylation-restricted sites are enriched for TF motifs containing CpGs, especially for those of NRF1. In fact, the TF NRF1 occupies several thousand additional sites in the unmethylated genome, resulting in increased transcription. Restoring de novo methyltransferase activity initiates remethylation at these sites and outcompetes NRF1 binding. This suggests that binding of DNA-methylationsensitive TFs relies on additional determinants to induce local hypomethylation. In support of this model, removal of neighbouring motifs in cis or of a TF in trans causes local hypermethylation and subsequent loss of NRF1 binding. This competition between DNA methylation and TFs in vivo reveals a case of cooperativity between TFs that acts indirectly via DNA methylation. Methylation removal by methylation-insensitive factors enables occupancy of methylation-sensitive factors, a principle that rationalizes hypomethylation of regulatory regions.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: DNase-seq (2 replicates) in mouse embryonic stem cells with (WT) and without DNA methylation (DNMT TKO). RNA-seq (3 replicates) in WT and DNMT TKO cells and in DNMT TKO cells after treatment with control siRNA or siRNA targeting Nrf1. H3K27ac ChIP-seq (2 replicates) in WT and DNMT TKO cells. NRF1 ChIP-seq (2 replicates) in WT and DNMT TKO cells, in WT upon culture in different conditions (adaptation to 2i and back to serum), upon transient overexpression of NRF1 and after differentiation into neuronal progenitor cells (NP). Whole-genome bisulfite sequencing in DNMT TKO cells and in WT upon culture in different conditions (adaptation to 2i and back to serum). NRF1 ChIP-seq (2 replicates) in human HMEC and HCC1954 cells.

first: Silvia

last: Domcke

middle: F

first: Dirk

last: Schübeler


title: The binding specificity and regulatory effect of WT and redesigned Puf2p

city: Stanford

first: Douglas

last: Porter

state: CA

address: 269 Campus Drive

department: Dermatology

institute: Stanford


title: ChIP-Seq experiments to analyse Rec8 and Zip1 localisation in Ctf19c mutants during meiotic prophase

city: Edinburgh

middle: Robert

state: Midlothian

address: 2.21 Michael Swann Building, Kings Buildings, Mayfield Road

department: Wellcome Centre for Cell Biology

country: United Kingdom

institute: University of Edinburgh


title: The binding specificity and regulatory effect of WT and redesigned Puf2p [CLIP-Seq]

summary: PUF proteins have become a leading scaffold for designing RNA-binding proteins to contact and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time. We show, by HITS-CLIP and PAR-CLIP, that S. cerevisiae Puf2p, a non-canonical PUF protein, binds more than 1000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We also perform CLIP-seq on truncations of the Puf2p, showing that its prion domain is dispensable for WT binding. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3’UTRs, such that the protein tracks with its sites, binds throughout the mRNA. We use RNA-seq to determine that R1 SNE Puf2p represses a novel RNA network.

overall_design: CLIP-seq was performed in BY4742 S. cerevisiae grown in log phase, and using 2 replicates of TAP-tagged proteins.

first: Yvonne

middle: Y

last: Koh

first: Brett

last: VanVeller

first: Ronald

middle: T

last: Raines

first: Marvin

last: Wickens


title: Inhibiting Fungal Multidrug Resistance by Disrupting an Activator-Mediator Interaction With the Small Molecule iKIX1

city: Charlestown

first: Joy

middle: Luz

last: Nishikawa

state: MA

address: Bldg 149, 13th Street

department: Cancer Center / Cell Biology

institute: Massachusetts General Hospital / Harvard Medical School

summary: Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. We previously identified an activator-targeted ~85 amino acid three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11 Mediator subunits in fungi. The Gal11 KIX domain is engaged by pleiotropic drug resistance transcription factor (Pdr1) orthologues, key regulators of the multidrug resistance (MDR) pathway in S. cerevisiae and in the clinically important human pathogen Candida glabrata. Drug-resistant clinical isolates of C. glabrata most commonly harbour point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway may represent a lynchpin in C. glabrata MDR. We have now carried out sequential biochemical and in vivo high-throughput screens to identify small molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation in both S. cerevisiae and C. glabrata and re-sensitizes drug-resistant C. glabrata to effective azole antifungal concentrations in vitro and in animal models for disseminated and urinary tract C. glabrata infection.

sample_organism:
Saccharomyces cerevisiae
Nakaseomyces glabratus

overall_design: Samples are generated in triplicate for four conditions (DMSO/vehicle-treated, iKIX1-treated, DMSO/vehicle and ketoconazole-treated. and iKIX1-ketoconazole treated) in both Saccharomyces cerevisiae and Candida glabrata

first: Anders

middle: M

last: Naar

first: F

last: Ji

first: Ruslan

middle: I

last: Sadreyev


title: ChIP-Seq experiment to analyse Zip1 localisation in the Ctf19c mutants during meiotic prophase.

first: Alastair

last: Kerr

summary: The goal of this experiment is to confirm whether Ctf19c mutants affect Zip1 localisation during meiotic prophase. To do so, diploid S. cerevisiae SK1 cells were constructed containing ndt80-d to arrest cells in prophase. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

overall_design: Diploid S. cerevisiae SK1 cells were constructed containing ndt80-d to arrest cells in prophase. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

first: Nadine

last: Vincenten

first: Bianka

last: Baying


title: RIP-seq of polyribosomal SLBP RNP

first: Michael

last: Whitfield

overall_design: Polyclonal α-SLBP antibody was used to immunoprecipitate SLBP RNP from polyribosomal fractions of HeLa S3  lysates. Mock immunoprecipitation were also performed and serve as a negative control.

first: Lionel

last: Brooks

middle: L


title: The binding specificity and regulatory effect of WT and redesigned Puf2p [RNA-Seq]

summary: PUF proteins have become a leading scaffold for designing RNA-binding proteins to contact and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time. We show, by HITS-CLIP and PAR-CLIP, that S. cerevisiae Puf2p, a non-canonical PUF protein, binds more than 1000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We also perform CLIP-seq on truncations of Puf2p, showing that its prion domain is dispensable for WT binding. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3’UTRs, such that the protein tracks with its sites and binds throughout the mRNA. We use RNA-seq to determine that R1 SNE Puf2p represses a novel RNA network.

overall_design: CLIP-seq was performed in BY4742 S. cerevisiae grown in log phase, and using 2 replicates of TAP-tagged proteins. RNA-seq was performed to determine the regulatory effect of WT or mutant Puf2p, using 4 replicates of the control (no Puf2p), 3 of WT Puf2p and 4 of R1 SNE Puf2p.


title: Embryonic transcription is controlled by maternally defined chromatin state

city: Nijmegen

first: Saartje

last: Hontelez

address: Geert Grooteplein 28

department: Molecular developmental biology

country: Netherlands

institute: Radboud University

summary: During development histone modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of embryonic epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3 and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300-recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences.

overall_design: We have performed ChIP-sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula (st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers (H3K4me1, p300), promoters (H3K4me3, H3K9ac), transcribed regions (H3K36me3, RNAPII) and repressed and heterochromatic domains (H3K27me3, H3K9me2, H3K9me3, H4K20me3). In addition we generated pre-MBT (st. 8) maps for three histone modifications (H3K4me3, H3K9ac, H3K27me3) and single-base resolution DNA methylome maps using whole genome bisulfite sequencing of blastula and gastrula (st. 9 and 10.5) embryos. To determine the maternal and zygotic contributions to chromatin state, we used alpha-amanitin to block embryonic transcription. Fertilised eggs were injected with 2.3 nl of 2.67 ng/ul alpha-amanitin and developed until the control embryos reached mid-gastrulation. Alpha-amanitin and control embryos were used for RNA-seq and ChIP-seq of RNAPII, H3K4me3, H3K27me3 and p300. For all ChIP-seq samples of the epigenome reference maps and RNAPII ChIP-seq samples of the α-amanitin experiments three biological replicates of different chromatin isolations of 45 embryos were pooled. Two biological replicates for H3K4me3 (α-amanitin injected: resp. 90 and 56 embryo equivalents (eeq); control: resp. 45 and 67 eeq), H3K27me3 (α-amanitin injected: resp. 90 and 180 eeq; control: resp. 45 and 202 eeq) and p300 (α-amanitin injected: resp. 112 and 56 eeq; control: resp. 112 and 67 eeq) ChIP-seq samples of the α-amanitin experiments were generated. For RNA-seq samples of the α-amanitin experiments RNA from 5 embryos from one biological replicate was isolated and depleted of ribosomal RNA

first: GertJan

middle: C

last: Veenstra


title: Elucidating microRNA regulatory networks using transcriptional, post-transcriptional and histone modification measurements

city: Cambridge

first: Sara

middle: J

last: Gosline

address: 77 Massachusetts Ave

department: Biological Engineering

institute: MIT


title: Identification of MEF2B, EBF1, and IL6R as chromosome bound targets of EBNA1 essential for EBV infected B-lymphocyte survival

first: Priyankara

last: Wickramasinghe

summary: EBNA1 is the EBV-encoded nuclear antigen required for viral episome maintenance during latency.  EBNA1 is a sequence specific DNA binding protein with high affinity binding sites for the viral genome, especially OriP.  EBNA1 can also bind sequence specifically to a large number of sites in the host cellular genome, but the function of these binding sites has remained elusive.  EBNA1 is also known to provide a host cell survival function, but the molecular mechanisms accounting for this function are not completely understood.  Here, we show by integrating ChIP-Seq and RNA-Seq with experimental validation that MEF2B, IL6R, and EBF1 are high confidence target genes of EBNA1 that are essential for viability of B-lymphocytes latently infected with EBV. We show that EBNA1 binds to ~1000 sites with many, but not all, universally bound in different cell types, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC).  We find that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity.  EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R,  EBF1, RNF145, POU2F1, KDM4C, FGR, EGFR, LAIR, CDC7, CD44, and IL17A. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation, and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1-depletion by decreasing EBV-positive cell growth and viability.  These findings indicate that EBNA1 binds to a large cohort of cellular genes important for cell viability, and implicates EBNA1 as a master coordinator of host cell gene expression important for enhanced survival of latently infected cells.

overall_design: Examination of EBNA1 binding in Raji, MutuI, LCL and C666-1 cells and EBNA1 knockdown effect on mRNA gene expression in LCL

first: Paul

last: Lieberman

first: Italo

last: Tempera


title: H3K27ac ChIP in Dicer KO/WT [ChIP-seq]

summary:
We performed ChIP-Seq of H3K27ac in duplicate in both WT and KO mesenchymal stem cells to evaluate global transcriptional changes between the new cells. We identified putative transcription factor binding sites using GEM v1.1 in K27ac data as well as in p
MicroRNAs (miRNAs) are small non-coding RNAs that regulates development and disease but induce only moderate repression of directs mRNA targets, suggesting that they coordinate with other modes ofs cellular regulation to effect large changes in gene expression. Ins this work we decouple direct effects of global miRNA loss froms transcriptional changes downstream in a pair of isogenic murines fibroblast cell lines with and without Dicer expression. Wes demonstrate how effects on direct miRNA targets are amplified bys transcription machinery through the construction of a network models that identifies specific transcription factors that cause changes ins mRNA expression upon Dicer loss. Through transcription factors over-expression, we delineate miRNA-mediated transcriptional programss and identify miRNA-mediated coherent and incoherent feed-forwards loops, suggesting a functional role of the interaction between miRNAss and transcription factors.   In total, our results indicate thats miRNAs tightly control transcription factors within a denses interconnected network to modulate gene expression.

overall_design: The experiment was designed to mimic the previously captured ChIP-Seq with two replicates in both WT and KO MSCs


title: mRNA-seq data of 14 days old Arabidopsis seedlings in Col-0 and At rz-1b At rz-1c double mutant

city: Norwich

first: Zhe

last: Wu

address: Colney

institute: John Innes Centre

summary: Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here we report the functions of two proteins containing RNA recognition motifs, At RZ-1B and At RZ-1C, in Arabidopsis. At RZ-1B and At RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C-termini. At RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA-binding activity of At RZ-1C with SR proteins through over-expression of the C-terminus of At RZ-1C conferred defective phenotypes similar to those observed in At rz-1b/At rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of At RZ-1B and At RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that At RZ-1C directly targeted FLC, promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of At RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

overall_design: mRNA-seq to look at the transcriptome and splicing differences between wild type and At rz-1b At rz-1c mutant of Arabidopsis thaliana

first: Lianfeng

last: Gu

first: Xiaofeng

last: Cao

first: Li-jia

last: Qu


title: Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming

city: Milan

first: Vania

last: Broccoli

address: Via Olgettina 58

department: Division of Neuroscience

country: Italy

institute: Ospedale San Raffaele

summary: Transplantation of GABAergic interneurons (INs) can sustain long-standing benefits in animal models of epilepsy and other neurological disorders. In a therapeutic perspective, a renewable source of functional GABAergic INs is needed. Here, we identified five factors (Foxg1, Sox2, Ascl1, Dlx5 and Lhx6) able to convert fibroblasts directly into induced GABAergic INs (iGABA-INs), displaying the molecular signature of telencephalic INs. The selected factors recapitulate in fibroblasts the activation of transcriptional networks required for the specification of GABAergic fate during telencephalon development. iGABA-INs exhibited progressively maturing firing patterns comparable to those of cortical INs, had synaptic currents and released GABA. Importantly, upon grafting in the hippocampus, iGABA-INs survived, matured and their optogenetic stimulation triggered GABAergic transmission and inhibited the activity of connected granule cells. The five factors also converted human cells into functional GABAergic neurons. These properties define iGABA-INs as a promising tool for disease modeling and cell-based therapeutic approaches.

overall_design: Comparison of iGABA-INs transcriptional profile with those of starting fibroblasts and GAD67-GFP+ cortical interneurons.

first: Gaia

last: Colasante

first: Gabriele

last: Lignani

first: Alicia

last: Rubio

first: Lucian

last: Medrihan

first: Latefa

last: Yekhlef

first: Alessandro

last: Sessa

first: Luca

last: Massimino

first: Serena

last: Giannelli

first: Silvio

last: Sacchetti

first: Massimiliano

last: Caiazzo

first: Damiana

last: Leo

first: Dimitra

last: Alexopoulou

first: Maria

last: Dell’Anno

first: Ernesto

last: Ciabatti

first: Marta

last: Orlando

first: Michele

last: Studer

first: Andreas

last: Dahl

first: Raul

middle: R

last: Gainetdinov

first: Stefano

last: Taverna

first: Fabio

last: Benfenati


title: RNA-seq of IL-4 stimulated human keratinocytes

city: Indianapolis

first: Mark

middle: H

last: Kaplan

state: IN

address: 1044 West Walnut St. Room 202

department: Pediatrics, and Microbiology and Immunology

institute: Indiana University

summary: In this study we aim to determine the role of IL-4/STAT6 in gene expression in human keratinocytes using RNA-sequencing approach. Human keratinocytes were cultured for 2 or 5 days with calcium chloride to induce terminal differentiation as determined by the expression of epidermal differentiation complex genes. The cells were then stimulated with IL-4 for 3 and 24 hours, or along the 5 days culture period.  We observed that IL-4 inhibits fully differentiation of keratinocytes, induces genes involved with production of inflammatory mediators, and reduces the healing capacity of human keratinocytes. Moreover, STAT6 controlled important genes involved with calcium binding, inflammation and epidermis development.

overall_design: Human keratinocytes were differentiated with calcium chloride for 2 days and incubated with media alone or 20ng/ml of recombinant human IL-4 for 3 and 24 hours. Human keratinocytes were differentiated with calcium chloride for 5 days with or wihout recombinant human IL-4 (20ng/ml). Keratinocytes transfected with control or STAT6 siRNA were differentiated with calcium chloride for 2 days and then stimulated with recombinant huma IL-4 for 24 hours.

first: Ana

last: Serezani


title: Genomic binding profiles of ERa in MCF-7 and T47D control or enhancer588-targeted cells

status: Public on Dec 14 2015

city: Tel Aviv

first: Ran

last: Elkon

address: Ramat Aviv

department: Human Genetics

country: Israel

institute: Tel Aviv University

summary: Estrogen Receptor a (ERa) bindning to DNA was profiled by ChIP-seq in MCF-7 and T47D cells transduced with either control sgRNA, or sgRNA targeting a specific enhancer region (enhancer588).

first: Gozde

last: Korkmaz

first: Rui

last: Lopes

first: Katja

last: nevedomskaya

first: Reuven

last: Agami


title: Single epicardial cell transcriptome sequencing identifies Caveolin-1 as an essential factor in zebrafish heart regeneration

first: Jingli

last: Cao

state: NY

department: Cell & Developmental Biology

institute: Weill Cornell Medical College

summary: By contrast with mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of spared cardiomyocytes.  The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies.  While it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored.  In this study, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21.  Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures.  We validated many new pan-epicardial and epicardial markers by alternative expression assays.  Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin-1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells.  Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration.  Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

overall_design: Deep sequencing of isolated single epicardial cells

first: Ben

middle: D

last: Cox

first: Kenneth

last: Poss


title: HSB-2 cells stably expressing LDB1 or mutant LDB1 proteins

city: Nashville

first: Yan

last: Guo

state: TN

address: 639 Preston Research Bld

institute: Vanderbilt Cancer Center

summary: LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated.  In this study we systematically dissected the LMO2/LDB1 binding interface to investigate the role of this interaction in T-cell leukemia.  Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif R320LITR required for LMO2 binding. Most strikingly, co-expression of full length, wild type LDB1 increased LMO2 steady state abundance, whereas co-expression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Raw gene expression data on HSB-2 cells is presented here.

overall_design: RNAseq were performed on HSB cell lines to examine their expression patterns

first: Utpal

last: Dave


title: Gene expression analysis to identify Mef2c/d target genes in B-cell progenitors

city: Hamburg

first: Julia

last: Herglotz

address: Martinistr. 52

institute: Heinrich-Pette-Institut

summary: The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms by which downstream programs are activated are incompletely understood. The preB-cell receptor (preBCR) is an important checkpoint of B-cell development and essential for a preB-cell to traverse into an immature B-cell. Here, we show that activation of Mef2 transcription factors by preBCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the preB-cell stage in mice deficient for Mef2c and Mef2d transcription factors and that preBCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the ERK5 mitogen activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.

overall_design: RNA-seq experiments were performed from Mef2c/d knockout proB-cells versus control cells to identify genes regulated by Klf2

first: Daniela

last: Indenbirken

first: Carol

last: Stocking


title: Gene expression analysis to identify target genes activated after preBCR signaling

overall_design: RNA-seq experiments were performed from Blnk-/- preB-cells with an integration of BLNK-ERt2 to identify genes regulated after preBCR signaling


title: Rec8 ChIP-Seq experiment to analyse cohesin localisation in the Ctf19c mutants during meiotic prophase.

first: Alastair

last: Kerr

summary: Ctf19c mutants were shown to specifically impair cohesin localisation at the pericentromere during mitosis. The goal of this experiment is to confirm whether this mutant has the same effect on cohesin localisation during meiotic prophase. To do so, diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

overall_design: Diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

first: Nadine

last: Vincenten

first: Bianka

last: Baying


title: Genome-wide mapping of Mef2c-bound sites in early B-cell progenitors

overall_design: ChIP-seq experiments were performed in the proB-cell line BMiFLT3(15-3) to identify Mef2c-bound sites in early B-cell progenitors.

first: Ludmilla

last: Unrau

first: Malik

last: Alawi


title: Essential control of early B-cell development by Mef2 transcription factors

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

overall_design: Refer to individual Series


title: SUMO and RNAPolII chromatin profiling in SUMOylation-competent and -deficient bone marrow derived dendritic cells unstimulated or stimulated with LPS

city: Paris

first: Adrien

last: Decque

address: 28 rue du Docteur Roux

institute: Institut Pasteur

summary:
Purpose: To determine SUMO1 and SUMO2 chromatin profile in a static and dynamic manner in BMDC before and after LPS stimulation, and to determine RNAPolII chromatin occupancy in sumoylation-deficient BMDC compared to wild-type cells.
Methods: SUMO1, SUMO2 and RNAPolII chromatin profiles were determined by sequencing BMDC chromatin immunoprecipitated with antibodies specific for SUMO1, SUMO2 and RNAPolII before and after LPS stimulation.
Results: We show dynamic  occupancy of three distal sites upstream of Ifnb1 gene by SUMO1 and SUMO2, as well as increased RNAPolII recruitment on selected genes.
Conclusions: SUMO acts as a regulator of inflammatory and anti-viral gene programs.

overall_design: A study of SUMO and RNAPolII chromatin profile in Bone Marrow derived Dendritic Cells.

first: Olivier

last: Joffre

first: Joao

middle: G

last: Magalhaes

first: Jack-Christophe

last: Cossec

first: Ronnie

last: Blecher-Gonen

first: Jacob

last: Seeler

first: Pierre

last: Lapaquette

first: Pierre-Emmanuel

last: Joubert

first: Matthew

last: Albert

first: Ido

last: Amit

first: Sebastian

last: Amigorena

first: Anne

last: Dejean


title: Sumoylation coordinates repression of inflammatory and anti-viral gene programs during innate sensing


title: Map of DnaC localization in the presence or absence of PcrA (PcrA depletion)

status: Public on Dec 13 2015

city: Seattle

first: Christopher

last: Merrikh

state: WA

address: 1705 NE Pacific St.

department: Microbiology Department

institute: University of Washington

summary: The replicative helicase, DnaC, was immunoprecipitated in cell extract prior to, or following conditional depletion of PcrA

sample_organism: Bacillus subtilis subsp. subtilis str. JH642

overall_design: The differential DnaC association at each genomic location was determined before/after PcrA depletion

first: Houra


title: H3K4me3 modification profiling of Huntingtons disease and control human post-mortem prefrontal cortex brain samples

city: Amherst

first: Lawrence

last: Schwartz

address: 211 Morrill Science Center, 611 North Pleasant Street

department: Department of Biology

institute: University of Massachusetts

summary: Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder resulting from expansion in the number of CAG repeats in the coding region of exon 1 of the Huntingtin (HTT) gene. One of the most widely studied chromatin modifications is trimethylated lysine 4 of histone 3 (H3K4me3). Here, we conducted the first comprehensive study of H3K4me3 ChIP-sequencing in neuronal chromatin from the prefrontal cortex of six HD cases and six non-neurologic controls. We examined two classes of H3K4me3 peaks; those located near transcription start sites (TSSs) (termed “proximal”) and those located distantly from TSSs (termed “distal”). We detected 2,830 differentially enriched H3K4me3 peaks between HD and controls, with 55% of them down regulated in HD.  We found an unexpected discordance between levels of H3K4me3 and mRNA, with H3K4me3 predominantly reduced in HD and mRNA predominantly increased in HD. Gene ontology (GO) term enrichment analysis of the genes associated with the proximally positioned peaks, revealed diverse biological process terms with organ morphogenesis and positive regulation of gene expression most frequently implicated. GO terms relating to transcription and the extracellular matrix were also observed.  Approximately 36% of the distal peaks co-localized to enhancer sites, with six transcription factors and chromatin remodelers differentially enriched in HD H3K4me3 distal peaks, including EZH2 and SUZ12, two core subunits of the polycomb repressive complex 2 (PRC2). Moreover, sequences repressed by polycomb in normal frontal cortex were significantly depleted in HD-enriched peaks, suggesting that H3K4me3 histone hypermethylation is linked to dysregulated polycomb activity in the HD neuronal epigenome.

overall_design: 12 H3K4me3 ChIP-seq libraries (6 Huntington's disease, 6 neurologically normal control), and 1 input DNA library (neurologically normal control)

first: Xianjun

last: Dong

first: Junko

last: Tsuji

first: Adam

last: Labadorf

first: Panos

last: Roussos

first: Jiang-Fan

last: Chen

first: Richard

last: Myers

first: Schahram

last: Akbarian

first: Zhiping

last: Weng


title: Map of DnaC localization in the presence or absence of PcrA (MycPcrA)

platform_organism: Bacillus subtilis subsp. subtilis str. JH642


title: Dynamically reorganized chromatin is the key for the reprogramming of somatic cells to pluripotent cells

city: Shanghai

first: Xiaobai

last: Zhang

address: 1239 Siping Road

institute: Tongji University

summary: We investigated genome-wide nucleosome coverage and histone methylation occupancies in somatic MEFs, intermediate pre-iPSCs and fully reprogrammed iPSCs. We found that nucleosome occupancies increased in promoter regions and decreased in intergenic regions in pre-iPSCs and then recover to an intermediate level in iPSCs. Nucleosomes in pre-iPSCs are much more phased than those in MEFs and iPSCs. Bivalent, active, repressive domains are cell type-specific and change dynamically during reprogramming. HCG and LCG promoters exhibit distinct nucleosome occupancy patterns and are dynamically marked by H3K4me3/H3K27me3 during reprogramming, correspondingly showing different gene activities. Surprisingly, Vitamin C promotes nucleosome reorganization from pre-iPSCs to iPSCs. CTCF binding sites change dynamically during reprogramming, though they share a conserved binding motif. Nucleosome occupies CTCF sites to a higher extent in pre-iPSCs than those in MEFs and iPSCs. Taken together, our study reveals that dynamic changes of nucleosome positioning and chromatin organization associated gene regulation during reprogramming.

overall_design: Examine dynamics of nucleosome positioning and histone modification profiles as well as gene expression regulation during somatic cell reprogramming

first: Kaimeng

last: Huang

first: Jiejun

last: Shi

first: Mingze

last: Yao

first: Jiannan

last: Lin

first: Jiao

last: Li

first: Liu

last: He

first: Huanhuan

first: Guang

first: Zhibin

last: Wang

first: Biliang

first: Jiekai

first: Guangjin

last: Pan

first: Cizhong

last: Jiang

first: Duanqing

last: Pei

first: Hongjie


title: Dicer WT/KO MSC RNA-Seq [total RNA]

first: Sara

last: Gosline

summary:
RNA-Seq performed on Dicer KO and WT murine mesenchymal stem cells from total RNA
MicroRNAs (miRNAs) are small non-coding RNAs that regulates development and disease but induce only moderate repression of directs mRNA targets, suggesting that they coordinate with other modes ofs cellular regulation to effect large changes in gene expression. Ins this work we decouple direct effects of global miRNA loss froms transcriptional changes downstream in a pair of isogenic murines fibroblast cell lines with and without Dicer expression. Wes demonstrate how effects on direct miRNA targets are amplified bys transcription machinery through the construction of a network models that identifies specific transcription factors that cause changes ins mRNA expression upon Dicer loss. Through transcription factors over-expression, we delineate miRNA-mediated transcriptional programss and identify miRNA-mediated coherent and incoherent feed-forwards loops, suggesting a functional role of the interaction between miRNAss and transcription factors.   In total, our results indicate thats miRNAs tightly control transcription factors within a denses interconnected network to modulate gene expression.

overall_design: Total RNA was analyzed from adult mesenchymal stem cells (immortalized monoclonal lines of murine MSCs) with and without Dicer (WT: Dicer f/f, KO: Dicer -/-), as well as from WT cells transfected with an empty vector or a vector containing Tead4, Sox9 or Pbx3 transcripts.


title: Circular RNAs are enriched in anucleate platelets and are a signature of transcriptome degradation

status: Public on Dec 12 2015

city: Newcastle Upon Tybe

first: Michael

last: Jackson

address: International Centre for Life

institute: Institute of Genetic Medicine

summary: In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-188 fold relative to nucleated tissues, and 14-26 fold relative to samples digested with RNAseR to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched ~13X in platelets relative to nucleated tissues, and identify 3162 genes significantly enriched for circRNAs including some where all RNAs appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in megakaryocytes, and that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost on aveage over 90% of their progenitor mRNAs, and that translation in platelets occurrs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artefacts.

overall_design:
A single rRNA depleted total RNA sample was sequenced. This together with 25 publicly available rRNA depleted total RNA samples (including 3 from platelets) were analysed using PTESFinder v 1 (http://sourceforge.net/projects/ptesfinder-v1/) to identify back-splice junctions, characteristic of circRNA transcripts. The contribution of circRNA producing exons was analysed on a gene by gene basis as follows: All circRNA transcripts identified in any sample were first pooled to define exons which can contribute to circRNA generation using custom scripts (available on request). For each sample, expression estimates (RPKMI) across all circRNA producing exons were computed for each locus using the total size of exons (in bp) and the read counts mapping to them. Similarly, total size and exonic read counts for exons for which no circRNA were detected in any sample were used to compute expression estimates (RPKME) for non-circRNA producing exons for each locus. Abundance ratios (RPKMI/RPKME and RPKMI/RPKMI+RPKME) were calculated and compared between Platelets and human tissues using Wilcoxon signed-rank test.

Please note that the '25sample_info_accn_no.txt' contains the accession numbers and tissue/cell type information for 25 samples analyzed together.

first: Abd

middle: A

last: Alhasan

first: Osagie

last: Izuogu

first: Haya

last: Al-Balool

first: Jannetta

last: Steyn

first: Amanda

last: Evans

first: Maria

last: Colzani

first: Cedric

last: Ghevaert

first: Joanne

last: Mountford

first: Lamin

last: Marenah

first: David

last: Elliott

first: Mauro

last: Santibanez-Koref


title: H3K27me3 histone modification in A549 cells

status: Public on Dec 11 2015

first: Isabelle

last: Janoueix-Lerosey

address: 26 rue d'Ulm

department: Inserm U830, Genetics and biology of cancers

institute: Institut Curie

summary: Examination of H3K27me3 histone modification in A549 cell line

overall_design: H3K27me3 ChiP-Seq


title: P21-activated kinase group II small compound inhibitor GNE-2861 perturbs estrogen receptor alpha signaling and restores tamoxifen-sensitivity in breast cancer cells

city: Huddinge

first: Karin

last: Dahlman-Wright

address: Blickagången 16

department: Department of Biosciences and Nutrition

country: Sweden

institute: Karolinska Institutet

summary: Estrogen receptor alpha (ERα) is highly expressed in most breast cancers. Consequently, ERα modulators, such as tamoxifen, are successful in breast cancer treatment, although tamoxifen resistance is commonly observed. While tamoxifen resistance may be caused by altered ERα signaling, the molecular mechanisms regulating ERα signaling and tamoxifen resistance are not entirely clear. Here, we found that PAK4 expression was consistently correlated to poor patient outcome in endocrine treated and tamoxifen-only treated breast cancer patients. Importantly, while PAK4 overexpression promoted tamoxifen resistance in MCF-7 human breast cancer cells, pharmacological treatment with a group II PAK (PAK4, 5, 6) inhibitor, GNE-2861, sensitized tamoxifen resistant MCF-7/LCC2 breast cancer cells to tamoxifen. Mechanistically, we identified a regulatory positive feedback loop, where ERα bound to the PAK4 gene, thereby promoting PAK4 expression, while PAK4 in turn stabilized the ERα protein, activated ERα transcriptional activity and ERα target gene expression. Further, PAK4 phosphorylated ERα-Ser305, a phosphorylation event needed for the PAK4 activation of ERα-dependent transcription. In conclusion, PAK4 may be a suitable target for perturbing ERα signaling and tamoxifen resistance in breast cancer patients.

overall_design: MCF7 cells were serum-starved for 72h and then treated with E2 for 45 min.

first: Ting

last: Zhuang

first: Jian

last: Zhu

first: Zhilun

last: Li

first: Julie

last: Lorent

first: Chunyan

last: Zhao

first: Staffan

last: Strömblad


title: The LIM homeodomain transcription factor Lhx2 is required for Müller glia development in the vertebrate retina.

city: Baltimore

first: Seth

last: Blackshaw

state: Maryland

address: 733 N Broadway

department: Neuroscience

institute: Johns Hopkins School of Medicine

summary:
Purpose: Müller glia are the only glial cell type produced by the neuroepithelial progenitor cells which generate the vertebrate retina.  Müller glia are required to maintain retinal homeostasis and support the survival of retinal neurons.  Furthermore, they function as an adult stem cell, mediating retinal regeneration among select vertebrate classes.  However, the mechanisms which regulate Müller development are poorly understood as considerable overlap exists in gene expression between retinal progenitor cells and differentiated Müller glia.  We investigate the functional role of the LIM homeodomain transcription factor Lhx2 in the specification and development of Müller glia in the mouse.
Methods: RNA-Seq was performed in collaboration with the Johns Hopkins School of Medicine Deep Sequencing and Microarray Core Facility.  Libraries were prepared using Illumina TruSeq RNA Sample kit (Illumina, San Diego, CA) following manufacturer’s recommended procedure.  The PCR amplified library was purified using RNAClean XP magnetic beads (Agencourt, Beverley, MA) and run out on a High Sensitivity DNA Chip (Agilent, Santa Clara, CA) for quality check.  We used STAR to align RNA-Seq reads onto Ensembl mouse genome GRCm38, release 72. To generate the stand attribute for alignments containing splice junctions, we used the outSAMstrandField intronMotif program. The spliced alignments without strand definition were removed. Number of reads mapped to exons was counted by htseq-count. Genes expressed at very low levels were omitted from further analysis.  Gene expression differences between wildtype and mutant samples, significance (p-value) and false discovery rate (FDR) were computed using the generalized linear models based EdgeR.
Results:  We observed a substantial reduction in expression of Notch pathway genes including Notch1, the Notch ligands Dll1 and Dll3, as well as gliogenic Notch effector genes such as Hes1, Hes5, Id1 and Sox8 and the Müller-gliogenic factor Rax.  We likewise observe a substantial reduction in expression of progenitor-specific genes such as Vsx2 and Fgf15.  Furthermore, we observed a decrease in the expression of early-onset glial markers such as Crym , Spon1, and Car2.

overall_design: Retinal mRNA profiles of post-natal day 0.5 (P0.5) Lhx2 wild type (N=3) and Lhx2lox/lox; Pdgfrα-Cre ΔcKO (N=3) mice were generated using Illumina TruSeq and analyzed with Agilent high sensitivity DNA analsis kit.

first: Jimmy

last: de Melo


title: Assessment of functional overlap between mouse Dux and human DUX4 in vitro and in vivo

city: Columbus

first: Scott

middle: Q

last: Harper

state: Ohio

address: 700 Children's Drive

department: Gene Therapy

institute: Nationwide Children's Hospital

summary: We report both DUX4 and Dux toxicity depend upon their ability to bind DNA and activate transcription. Chromatin immunoprecipitation of V5 epitope tagged human DUX4 and mouse Dux was performed in human myoblasts was analyzed using ChIP-Seq to identify their subsequent binding sites. We found that DUX4 and Dux bind 4-8% of identical sequences, while majority of the binding sites are unique to either DUX4 or Dux. Although small, this overlap could be due to their conserved abilioty to regualte primordial pathways that were essential for life and therefore maintained in both proteins despite their separate evolutionary paths.

overall_design: We performed ChIP-Seq analysis of human myoblasts transfected with plasmids encoding either epitope tagged human DUX4 (1 sample) and mouse Dux (1 sample). Illumina sequencing libraries were prepared from the ChIP and Input DNA, then resulting DNA libraries were quantified and sequenced and aligned to the human genome (hg19).

first: Jocelyn

middle: O

last: Eidahl

first: Carlee

last: Giesige


title: Aedes aegypti Cry11Aa intoxication curve transcriptome

city: Gainesville

first: Pablo

middle: Emiliano

last: Canton

state: Florida

address: Natural Area Dr.

department: Entomology and Nematology

institute: University of Florida

summary: Transcriptomic analysis of mosquito larvae exposed to LC50 of Cry11Aa toxin from Bti at different times was conducted to determine defense response pathways in order to better understand the toxin's mode of action and identify possible cellular targets to enhance toxin activity.

overall_design: 4 replicates of 5 intoxication time points and 3 replicates of 2 control timepoints were sequenced on an Illumina platform, analyzed for differential expression with two packages. Read data can be accessed through PRJNA301038 and SRP065731

middle: E

first: Alejandra

last: Bravo

first: Mario

last: Soberón

first: Sarjeet

middle: S

last: Gill

first: Angeles

last: Cancino-Rodezno


title: Gene expression analysis to identify Klf2 target genes in B-cell progenitors [+Klf2]

first: Julia

last: Herglotz

overall_design: RNA-seq experiments were performed from Klf2 overexpressing BMiFLT3 (15-3) cells to identify genes regulated by Klf2

first: Daniela

last: Indenbirken

first: Carol

last: Stocking


title: Downregulation of LATS kinases alters p53 to promote cell migration

city: Rehovot

first: Noa

last: Bossel Ben-Moshe

address: Hertzl Street

institute: Weizmann

summary: p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in non-transformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-?B subunit. Moreover, it partly shifts p53’s conformation and transcriptional output towards a state resembling cancer-associated p53 mutants, and endow p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently downregulated in breast cancer; we propose that such downregulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.

overall_design: MCF10A cells transfected with siRNA against LATS1/2 alone, p53 alone or LATS1/2 and p53 together. Two independent MCF10A batches provided biological replicates

last: Furth

middle: B

last: Ben-Moshe

first: Yair

last: Pozniak

first: Ziv

last: Porat

first: Tamar

last: Geiger

first: Eytan

last: Domany

first: Yael

last: Aylon

first: Moshe

last: Oren


title: Perinuclear anchoring of H3K9-methylated chromatin stabilizes induced cell fate

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute


title: DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

status: Public on Dec 21 2015

city: New York

first: Wendy

last: Huang

address: 540 First Ave Skirball 2-14

institute: NYU School of Medicine


title: mRNA Sequencing of skeletal muscle genes in wildtype and BCATm (BCAT2) KO mice

city: Hershey

first: Yuka

last: Imamura Kawasawa

address: 500 University Dr.

department: College of Medicine, Pharmacology

institute: Penn State University

summary: Consumption of a protein containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), that interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wildtype mice were compared using Next Generation RNA-Sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1486/~39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis (eIF2, mTOR, eIF4 and p70S6K pathways including 40S and 60S ribosomal proteins), protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy and cell death) were up-regulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways were observed in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids and BCAAs were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wildtype mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes.

overall_design: Comparison of wildtype and BCATm KO gastrocnemius biological replicates

first: Christopher

last: Lynch

first: Scot

last: Kimbal

first: Yuping

last: Xu

first: Anna

last: Salzberg

last: Kawasawa


title: H3K4me3 chromatin profiling in SUMOylation-competent and -deficient bone marrow derived dendritic cells unstimulated or stimulated with LPS

first: Adrien

last: Decque

summary:
Purpose: To determine H3K4me3 chromatin profile in UBC9WT and UBC9KO BMDC before and after LPS stimulation.
Methods: H3K4me3 chromatin profile was determined by sequencing UBC9 WT and UBC9 KO BMDC chromatin immunoprecipitated with antibody specific for H3K4me3.
Results: We show differential chromatin profile for H3K4me3 on the ifnb1 locus between UBC9 KO cells and UBC9 WT.
Conclusions: Loss of SUMOylation causes a deregulation of ifnb1 transcription activation mark.

overall_design: A study of H3K4me3 chromatin profile in UBC9 WT and UBC9 KO Bone Marrow derived Dendritic Cells.

first: Olivier

last: Joffre

first: Joao

last: Magalhaes

first: Jack-Christophe

last: Cossec

first: Ronnie

last: Blecher-Gonen

first: Jacob

last: Seeler

first: Pierre

last: Lapaquette

first: Pierre-Emmanuel

last: Joubert

first: Matthew

last: Albert

first: Ido

last: Amit

first: Sebastian

last: Amigorena

first: Anne

last: Dejean


title: Tet1 plays an essential oncogenic role in MLL-rearranged leukemia

city: Atlanta

first: Keith

last: Szulwach

state: GA

address: 615 Michael Street, Whitehead Suite 301

institute: Emory University

summary: TET1, the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), was first identified as a partner gene in MLL-rearranged leukemia, but its definitive pathological role in leukemia is unclear. The down-regulation of all three TET genes and loss-of-function mutations of TET2 have been frequently observed in various cancers, and it was thought that they all play tumor-suppressor roles in tumorigenesis. Here we show that TET1 is likely a direct target of MLL and significantly up-regulated in MLL-rearranged leukemia, associated with an increased level of 5hmC. Our further in vitro and in vivo studies demonstrate that Tet1 plays an indispensable oncogenic role in MLL-rearranged leukemia, through cooperating with MLL fusion proteins in regulating their co-targets including the Hoxa/Meis1/Pbx3/Flt3 genes. Our data delineate a MLL-fusion/Tet1/Hoxa/Meis1/Pbx3/Flt3 signaling axis in MLL-rearranged leukemia, and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.

overall_design: We report genome-wide 5hmC enrichment profiles and RNA-Seq gene expression in MLL-AF9 transformed and control mouse bone marrow mononuclear cells. These 5hmC profiles are derived from selctive chemical labeling and enrichment of 5hmC containing genomic DNA fragments, while the RNA-Seq expression profiles are generated from polyA enriched RNA


title: Genome wide identification of enhancer regions in the  retrosplenial cortex of the prairie vole, Microtus ochrogaster.

city: Austin

first: Steven

last: Phelps

state: TX - Texas

address: 34 Patterson Labs, 2401 Speedway

department: Integrative biology

institute: University of Texas at Austin

summary: We use ChIP-seq targeting histone 3 lysine 4 mono-methylation (H3K4me1)  to identify putative enhancer sites genome-wide, in the retrosplenial cortex of adult prairie vole males.

overall_design: ChIP samples were generated by targeting a known enhancer mark (H3K4me1) in chromatin extracted from the retrosplenial cortex of 8 males. Illumina libraries were prepared from ChIP and INPUT DNA and sequenced on Illimuna HiSeq 2500 platform.

first: Mariam

last: Okhovat


title: Gene expression analysis to identify Klf2 target genes in B-cell progenitors [Klf2_KO]

first: Julia

last: Herglotz

address: Martinistr. 52

institute: Heinrich-Pette-Institut

summary: The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms by which downstream programs are activated are incompletely understood. The preB-cell receptor (preBCR) is an important checkpoint of B-cell development and essential for a preB-cell to traverse into an immature B-cell. Here, we show that activation of Mef2 transcription factors by preBCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the preB-cell stage in mice deficient for Mef2c and Mef2d transcription factors and that preBCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the ERK5 mitogen activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.

overall_design: RNA-seq experiments were performed from Klf2 knockout proB-cells versus control cells to identify genes regulated by Klf2

first: Daniela

last: Indenbirken

first: Carol

last: Stocking


title: Dual RNA-seq – rRNA depletion establishment

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

institute: ZB MED - Information Centre for Life Sciences

summary: Bacteria possess many small noncoding RNAs whose regulatory roles in pathogenesis are little understood due to a paucity of macroscopic phenotypes in standard virulence assays. Here, we use a novel Dual RNA-seq approach for a single-step simultaneous RNA profiling in both pathogen and host to reveal molecular phenotypes of sRNAs during infection with Salmonella Typhimurium. We identify a new PhoP/Q-activated small RNA which upon bacterial internalization acts to temporally control the expression of both, invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity is shown to adjust the human response to replicating Salmonella, and have a pervasive impact on host RNA expression both inside and outside protein-coding regions including infection-specific alterations of an array of long noncoding RNAs. Our study provides a paradigm for a comprehensive RNA-based analysis of intracellular bacterial pathogens without their physical purification from a host and a new discovery route for hidden functions of pathogen genes.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: rRNA depletion establishment

first: Alexander

last: Westermann

middle: U

first: Fabian

last: Amman

first: Lars

last: Barquist

first: Leon

middle: N

last: Schulte

first: Yanjie

last: Chao

first: Lydia

last: Müller

first: Richard

last: Reinhardt

first: Peter

middle: F

last: Stadler

first: Jörg

last: Vogel


title: Dual RNA-seq – Comparative RNA-seq (in vitro medium shift experiment)

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium


title: Dual RNA-seq –  Dual RNA-seq of further sRNA mutants

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: Dual RNA-seq of further sRNA mutants


title: Dual RNA-seq – Host response to pinT*

overall_design: Host response to pinT*


title: Dual RNA-seq – Comparative Dual RNA-seq in pig macrophages

overall_design: Comparative Dual RNA-seq in pig macropahges


title: RNA-Seq of human astrocytes

last: Sloan

address: 615 Michael Street, Whitehead Building Room 375

summary: Astrocytes were purified from fetal and adult human brain tissue using an immunopanning method with the HepaCAM antibody. Samples were taken from otherwise 'healthy' pieces of tissue, unless otherwise specified.

overall_design: 6 fetal astrocyte samples, 12 adult astrocyte samples, 8 GBM or sclerotic hippocampal samples, 4 whole human cortex samples, 4 adult mouse astrocyte samples, and 11 human samples of other purified CNS cell types

first: Ye

last: Zhang


title: CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription

city: Farmington

first: Oscar

middle: J.

last: Luo

state: CT

address: 10 Discovery Dr

institute: The Jackson Laboratory for Genomic Medicine

summary: Spatial genome organization and its effect on transcription remains a fundamental biological question. We applied an advanced ChIA-PET strategy to comprehensively map higher-order chromosome folding and chromatin interactions mediated by CTCF and RNAPII with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes towards CTCF-foci and forming RNAPII foci for coordinated transcription. Furthermore, allelic-variants linked to disease susceptibility are found to impact chromatin topology and transcriptional regulation. Importantly, 3D-genome simulation suggested a model of chromatin folding around chromosomal axes, where CTCF involves in defining the interface between condensed and open compartments for structural regulation. Our new 3D-genome strategy thus provides novel insights to explore the topological mechanism of human variations and diseases.

overall_design: Investigation of chromatin organization and function by CTCF ChIA-PET

first: Zhonghui

last: Tang

first: Xingwang

last: Li

first: Yijun

last: Ruan


title: Parallel single-cell bisulfite- and RNA-sequencing link transcriptional and epigenetic heterogeneity

status: Public on Dec 24 2015

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

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

overall_design:
Refer to individual Series

Supplementary file GSE74535_source_code.tar.gz: Intermediate data files required to reproduce the results with the provided source code (https://github.com/PMBio/scMT-seq).


title: Dual RNA-seq – High-resolution comparative Dual RNA-seq time-course

last: Förstner

middle: J


title: Dual RNA-seq – Pulse-expression of pinT

overall_design: Pulse-expression of pinT

last: Westermann


title: Genome-wide analysis reveals NRP1 as a critical HIF1-E2F7 target gene in the regulation of motorneuron guidance in vivo

city: Utrecht

first: Michal

last: Mokry

address: Lundlaan 6

institute: Wilhelmina Children's Hospital, University Medical Center Utrecht

summary: HIF1 is essential for regulation of the transcriptional response to hypoxia. Recently we showed that the transcriptional repressors E2F7 and E2F8 interact and transcriptionally cooperate with HIF1. Here we further explored this cooperation by performing genome-wide analysis, screening for novel HIF1-E2F7 targets. We show that specifically E2F7 is induced in hypoxia by HIF1. Furthermore, chip-sequencing for E2F7 and HIF1 revealed a large number of common targets of which a subset was also regulated by the complex as examined by microarray analysis. Our data show that the HIF1-E2F7 complex can function both as a repressor or activator. Notably, we identify neuropilin 1 (NRP1) as a novel HIF1-E2F7 target, which is repressed by HIF1-E2F7 in vitro and during zebrafish development, depending on E2F-binding sites present in the NRP1 promoter. In addition we show that regulation of NRP1 by the HIF1-E2F7 complex is required for normal axon guidance of spinal motorneurons in vivo.

overall_design: ChIP-seq analysis of HIF1a and E2F7 binding

first: A

last: de Bruin

first: PW

last: Cornelissen

first: BC

last: Kirchmaier

first: M

first: E

last: Iich

last: Nirmala

first: KH

last: Liang

last: Végh

first: KT

last: Scholman

first: MJ

last: Groot Koerkamp

first: FC

last: Holstege

last: Cuppen

first: J

last: den Hertog

first: S

last: Schulte-Merker

first: WJ

last: Bakker


title: Dual RNA-seq – Pilot Dual RNA-seq

first: Konrad

last: Förstner

overall_design: Pilot Dual RNA-seq: Infection of HeLa-S3 cells with wild-type Salmonella; 2 time points (4 h, 24 h p.i.; each sorted into invaded host cells [GFP+] and non-infected bystanders [GFP-]) and the respective human (4 h mock, 24 h mock) or bacterial reference controls (0 h LB,  0 h input), respectively. Three biological replicates were taken.

first: Alexander

first: Fabian

last: Amman

first: Lars

last: Barquis

first: Leon

last: Schulte

first: Yanjie

last: Chao

first: Lydia

last: Müller

first: Richard

last: Reinhardt

first: Peter

last: Stadler

first: Jörg

last: Vogel


title: RNA-seq for monitoring expression changes in absence of chromatin anchoring

first: Dimos

last: Gaidatzis

summary: RNA-seq for monitoring expression levels in mutants that do not anchor chromatin at the nuclear periphery.

overall_design: RNA-seq of depleted rRNA samples of early embryo extracts for three different genotypes: wild-type, cec-4_delta and met-2 set-25_delta_delta, in two independent biological replicas

first: Adriana

last: Gonzalez-Sandoval


title: RNA sequencing of a C57BL/6J-DBA/2J (B6.D2) congenic mouse and C57BL/6J (B6) wild-type littermates reveals cis and trans differences in gene expression

city: Boston

first: Camron

middle: D.

last: Bryant

address: 72 E. Concord St. R-604

department: Pharmacology & Experimental Therapeutics

institute: Boston University School of Medicine

summary: We previously utilized interval-specific congenic lines derived from C57BL/6J (B6) and DBA/2J  (D2) alleles to fine map a quantitative trait locus (QTL) influencing methamphetamine (MA)-  induced locomotor activity. We identified a 0.23 MB critical interval on chromosome 11  containing only two protein coding genes, Rufy1 and Hnrnph1. Notably, Rufy1 contains three  missense SNPs and Hnrnph1 contains 1 SNP near the 5’ UTR. In an effort to identify the molecular  mechanisms that bridge genetic variation with behavior, we conducted transcriptome analysis  via mRNA sequencing (RNA-seq) in a B6.D2 congenic line (chr.11: 50-60 Mb) that captures the  QTL. There was an overrepresentation of cis-regulated, differentially expressed genes within  the congenic interval (4 out of 92 differentially expressed genes; FDR < 0.05) and widespread  genomic regulation on all autosomes.

overall_design: For this study, 3mm punches from both striatal hemispheres were collected and pooled from 16 mice (8 controls and 8 B6.D2 congenics), and RNA was extracted and prepared for cDNA library preparation using the Illumina TruSeq (oligo-dT; 50bp single-end reads). Samples 1-16, consisting of alternating B6 and B6.D2 congenics were run in four lanes total, with samples 1-8 run in duplicate across lanes 5 and 6 and samples 9-16 acorss lanes 7 and 8, respectively. Raw samples provided are labeled with the "CB-" prefix, followed by the sample number and the lane that it was run in is described at the tail end as "_L00#" with # being either lane 5, 6, 7 or 8. In this study, all odd samples (e.g. CB-1,3,5 etc.) are C57BL/6J littermate controls, while all even samples are B6.D2 congenics. Note that the lane 7 replicate for Sample CB-13 (CB-13_L007.fastq.gz) was not processed due to technical difficulties with the file.

first: Neema

last: Yazdani

last: D. Bryant

first: W

last: Johnson

first: Ying

last: Shen


title: LEM-2 ChIP-seq to study chromatin anchoring

summary: Chromatin Immunoprecipitation against the nuclear envelope protein LEM-2 to detect association to the nuclear periphery. LEM-2 ChIP was performed on early embryo fixed extracts for wild-type and two mutants that have altered position of a perinuclear chromatin reporter.

overall_design: LEM-2 ChIP on early embryo chromatin extracts of three different genotypes: wild-type, met-2 set-25_delta_delta and cec-4_delta, in two independent biological replicas


title: Super-Enhancer-mediated Control of Liver Fibrosis by BET Bromodomain Proteins

city: La Jolla

first: Ruth

middle: T

last: Yu

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute

summary: Here, we investigate the role of enhancers in myofibroblasts, a cell type that dominates the pathogenesis and progression of tissue fibrosis. We reveal that bromodomain and extra-terminal family members (BETs), an important group of epigenetic readers, are critical for super-enhancer-mediated pro-fibrotic gene expression in hepatic stellate cells (HSCs, lipid-containing liver-specific pericytes), upon activation during liver fibrogenesis give rise to myofibroblasts2-4. We observe significantly enriched localization of BETs to hundreds of super-enhancers associated with genes involved in multiple pro-fibrotic pathways. This unique loading pattern consequentially serves as a molecular mechanism by which BETs modulate pro-fibrotic gene expression in myofibroblasts. Strikingly, suppression of BET-enhancer interaction using small-molecule inhibitors such as JQ1 dramatically blocks activation of HSCs into myofibroblasts and significantly compromises the proliferation of activated HSCs.

overall_design: Identification of BRD2, BRD3, BRD4, PolII, PolIIs2p and PolIIs5p binding sites in human stellate LX2 cells that were treated with DMSO (0.1%) or JQ1 (500nM) for 16 hrs.

first: Ning

last: Ding

first: Ronald

middle: M

last: Evans


title: RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

first: Robert

last: Roeder

address: 1230 York Avenue

institute: The Rockefeller University

summary: Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.

overall_design: Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.

first: Ming

first: Wenjing

last: Yang

first: Ting

last: Ni

first: Zhanyun

last: Tang

first: Tomoyoshi

last: Nakadai

first: Jun

last: Zhu

middle: G


title: Methylated DNA immunoprecipitation-sequencing of HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)

city: Jouy-en-Josas

first: Helene

last: Bierne

address: Domaine de Vilvert, bat 442, INRA Micalis

department: Micalis Institute

institute: INRAE - University PARIS SACLAY

summary: we report the partial methylome (CG-rich regions) of HEK293 cells and HEK293 cells over-expressing the BAHD1 gene (HEK-BAHD1)

overall_design: We used MEDIP-seq to identify genomic regions differentially methylated upon overexpression of the chromatin repressor BAHD1 in HEK293 cells.

first: Emanuele

last: Libertini

first: Anupam

last: Paliwal

first: Caroline

last: Proux

first: Jean-Yves

last: Coppee


title: whole-genome bisulfite sequencing (BS-seq) of HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)

summary: Comparison of methylome of HEK293-CT cells and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)

overall_design: We used BS-seq to identify genomic regions differentially methylated upon overexpression of the chromatin repressor BAHD1 in HEK293 cells.

first: Stefan

last: Beck


title: RNAseq from neonatal mouse hindlimb muscles

status: Public on Dec 10 2015

first: Jinmo

last: Gu

state: OH

address: 460 W 12th Ave

department: CCC

institute: The Ohio State University

summary: During neonatal development, skeletal muscle grows dramatically by myonuclei accretion to existing fibers and hypertophic growth of fibers with protein synthesis.

overall_design: To understand molecular mechanism underlying neonatal muscle growth, we used RNAseq to profile the global program of gene expressions especially involved in myoblast fusion, migration, and muscle fiber growth by itself. We used two biological replicates for each time point.

first: Ayse

last: Yilmaz

first: Sandya

last: Liyanarachchi

first: Denis

middle: C

last: Guttridge


title: Gonadal transcriptome analysis in sterile double haploid Japanese flounder

city: qinhuangdao

first: xiaoyan

last: zhang

address: kangle road

institute: Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences

summary: We used the Illumina sequencing platform to analyze the transcriptome of sterile female DH Japanese flounder in order to identify major genes that cause sterility and to provide a molecular basis for an intensive study of gonadal development in teleosts. Through sequencing, assembly, and annotation,

overall_design: Three sterile female japanese flounders were control samples and three fertile ones were references samples

first: Xiao-Yan

last: Zhang

first: Ji-Lun

last: Hou

first: Chen

last: Dai


title: Chromatin immunoprecipitation-sequencing of HEK293 cells (HEK293-HPT-blue) and HEK293 cells stably over-expressing the BAHD1 gene (HEK293-HPT-BAHD1)

summary: We report the genomic occupancy of the BAHD1 protein in HEK293 cells over-expressing the BAHD1 gene (HEK293-HPT-BAHD1)

overall_design: We used Native ChIP-seq to identify DNA regions bound to BAHD1 (native ChIP involves use of native chromatin before precipitation of immune complexes, without formaldehyde crosslinking of proteins with nucleic acids)

first: Alice

last: Lebreton

first: Goran

last: Lakisic


title: HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)

overall_design: Refer to individual Series


title: A dynamic unfolded protein response contributes to the control of cortical neurogenesis

city: Münster

first: Danny

last: Nedialkova

address: Von-Esmarch-Str. 54

department: Max Planck Research Group for RNA Biology

institute: Max Planck Institute for Molecular Biomedicine

summary: The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs) whose asymmetric division give birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Our data suggest that interfering with codon translation speed triggers endoplasmic reticulum stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a hitherto unrecognized contribution of UPR to cell fate acquisition during mammalian brain development.

overall_design: Ribosome profiling and RNA-Seq of forebrains from E14.5 mouse embryos from wild type animals and mutants carrying a conditional knockout of ELP3 in cortical progenitors

middle: D


title: Dual RNA-seq – sRNA profiling in various cell types

first: Konrad

last: Förstner

overall_design: sRNA profiling in various cell types

first: Alexander

last: Westermann

first: Fabian

last: Amman

first: Lars

last: Barquist

first: Leon

last: Schulte

first: Yanjie

last: Chao

first: Lydia

last: Müller

first: Richard

last: Reinhardt

first: Peter

last: Stadler

first: Jörg

last: Vogel


title: Stem cells rejuvinate the schistosome host-parasite interface

city: Dallas

first: James

last: Collins

state: TX

address: 6001 Forest Park

department: Pharmacology

institute: UT Southwestern

summary: We report the transcriptional effects of long-term stem cell depletion in Schistosoma mansoni

overall_design: Examination of stem cell depltion by irradiation, and RNAi

first: Phillip

middle: A

last: Newmark


title: Limiting cholesterol biosynthetic flux engages type I IFN signaling in a STING-dependent manner

city: Los Angeles

first: Steven

last: Bensinger

address: 615 Charles E Young Dr

department: MIMG

institute: UCLA

summary: Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling rapidly shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol, and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

overall_design: shRNA to SREBF1 (shSREBP1) or SREBF2 (shSREBP2) were stably introduced via 3rd generation lentivirus into human THP1 monocytic cells under puromycin selection. Non-targeting shRNA scramble was used for a control (shControl). shControl, shSREBP1 and shSREBP2 modified cell types were analyzed by RNA-seq in duplicate.

first: Autumn

last: York

first: Kevin

last: Williams

first: Joseph

last: Argus

first: Quan

last: Zhu

first: Laurent

last: Vergnes

first: Elizabth

last: Gray

first: Anjie

last: Zhen

first: Nicholas

last: Wu

first: Gubpreet

last: Brar

first: Douglas

last: Yamada

first: Cameron

last: Cunningham

first: Moses

middle: Q

last: Wilks

first: David

last: Casero

first: Amy

last: Yu

last: Brooks

first: Ren

last: Sun

first: Scott

last: Kitchen

first: Ting-Ting

first: Karen

last: Reue

first: Daniel

last: Stetson


title: Gene expression profiling of neurons with Rbfox1 and Rbfox3 knockdown and rescue with cytoplasmic or nuclear Rbfox1 isoform [RNA-seq]

city: LOS ANGELES

first: Chia-ho

last: Lin

address: 6577 MRL. 675 CHARLES E YOUNG DR. SOUTH

summary: Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes induced by knockdown, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that in nascent RNA Rbfox1 bound predominantly to introns, while cytoplasmic Rbox1 bound to 3' UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and overlapped significantly with miRNA binding sites. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease. In this data set, we included the data from RNA-seq experiments.

overall_design: We performed RNA-seq to profile gene expression and splicing changes. The expression levels of Rbfox1 and Rbfox3 in cultured mouse hippocampal neurons were reduced by siRNAs. The reduction of Rbfox1 and 3 was rescued by expression of cytoplasmic or nuclear Rbfox1 splice isoform. The gene expression and splicing profiles were compared between different treatments. Eight samples were analyzed.

first: Ji-Ann

last: Lee

first: Chia-Ho

first: Kelsey

last: Martin

middle: L

last: Black


title: The histone chaperone CAF-1 safeguards somatic cell identity during transcription factor-induced reprogramming (sequencing)

first: Peter

last: Park

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary:
During development, specialized cell lineages are generated through the establishment of cell type-specific transcriptional patterns and epigenetic programs. However, the precise mechanisms and regulators that maintain these specialized cell states remain largely elusive. To identify molecules that safeguard somatic cell identity, we performed two comprehensive RNAi screens targeting known and predicted chromatin regulators during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation, DNA methylation and heterochromatin maintenance. Suppression of CAF-1 increased reprogramming efficiencies by several orders of magnitude and generated iPSCs two to three times faster compared to controls without affecting cell proliferation. We demonstrate that suppression of CAF-1 leads to a more accessible chromatin structure specifically at enhancer elements early during reprogramming. These changes were accompanied by increased binding of the reprogramming factor Sox2 to ESC-specific regulatory elements and earlier activation of pluripotency-associated genes. Notably, suppression of CAF-1 also enhanced iPSC formation from blood progenitors as well as the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as an unanticipated regulator of somatic cell identity and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Keywords: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Chromatin accessibility and Sox2 bindings in CAF-1 knockdown and Renilla control during early OKSM reprogramming by high throughput sequencing

first: Konrad

last: Hochedlinger

first: Sihem

last: Cheloufi


title: Cytoplasmic Rbfox1 regulates the neuronal expression of synaptic and autism-related genes

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


title: DNA elimination analysis of Tetrahymena cells defective in heterochromatin body formation

city: Montpellier

first: Kazufumi

last: Mochizuki

address: 141 rue de la Cardonille

institute: Institute of Human Genetics, CNRS

summary: In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the novel heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1 (HP1)-like protein Pdd1p. Through the identification and mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore suggest that heterochromatin bodies are assembled by the Pdd1p-RNA interaction. Jub1p and Pdd1p dephosphorylation are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body of itself plays an essential role in DNA elimination.

overall_design: Micronuclei (MICs) and new macronuclei (MACs) of exconjugants were isolated from different mutants at 36 hpm, and the genomic DNA was analyzed by high-throughput sequencing.

first: Kensuke

last: Kataoka