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ELAV ChIP-seq in Drosophila melanogaster embryos

ABSTRACT: Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes, such as stem cell differentiation and cancer. A particularly dramatic form of APA has been documented in the developing nervous system of flies and mammals, whereby a variety of neurogenic genes undergo coordinate extension of their 3’ UTRs. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation (poly(A)) sites, thereby fostering the formation of 3’ extensions that can reach 12 kb in length. Here, we present evidence that paused Pol II plays an important role in the selective recruitment of ELAV to elongated genes. Replacing native promoters of elongated genes with heterologous promoters blocks normal 3’ extension in the nervous system, while native promoters can induce 3’ extension in ectopic tissues expressing ELAV. Computational analyses suggest that the promoter regions of elongated genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ELAV ChIP-Seq assays indicate pervasive binding to the promoter regions of extended genes. Our study provides the first evidence for a regulatory link between promoter-proximal pausing and APA. ELAV ChIP-Seq assays were conducted with nuclei obtained from 6-8 hr and 10-12 hr embryos

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Wei Zhang

PROVIDER: E-GEOD-63323 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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ELAV ChIP-seq in Drosophila melanogaster embryos

Project description:Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes, such as stem cell differentiation and cancer. A particularly dramatic form of APA has been documented in the developing nervous system of flies and mammals, whereby a variety of neurogenic genes undergo coordinate extension of their 3’ UTRs. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation (poly(A)) sites, thereby fostering the formation of 3’ extensions that can reach 12 kb in length. Here, we present evidence that paused Pol II plays an important role in the selective recruitment of ELAV to elongated genes. Replacing native promoters of elongated genes with heterologous promoters blocks normal 3’ extension in the nervous system, while native promoters can induce 3’ extension in ectopic tissues expressing ELAV. Computational analyses suggest that the promoter regions of elongated genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ELAV ChIP-Seq assays indicate pervasive binding to the promoter regions of extended genes. Our study provides the first evidence for a regulatory link between promoter-proximal pausing and APA.

2014-11-21 | GSE63323 | GEO

ELAV proteomics after brain ischemia and reperfusion in the rat

Project description:We investigated mRNA regulatory proteins of the ELAV family following 10 min global brain ischemia and 8 hrs reperfusion (8R) or non-ischemic controls (NIC) in rat hippocampal CA1 and CA3. There were three main experiments: (1) Shot-gun proteomics of polysome pellets from NIC and 8R CA1 and CA3, (2) Shot-gun proteomics of ELAV immunoprecipitate eluents from NIC and 8R CA1 and CA3, and (3) Proteomics of ELAV antisera-reactive bands excised from nitrocellulose following ELAV Western blot.

2016-07-07 | PXD004078 | Pride

The E3 Ligase CUL3 physically interacts with TANGO10 protein in vivo

Project description:List of protein scores for putative TANGO10 interactors from mass spectrometry analysis of FLAG-tagged TANGO10 using either elav-GAL4 or tim-GAL4 at both ZT10 and ZT 22, as determined using Proteome Discoverer software (ThermoFisher). Fly heads from GAL4 heterozygotes were used as controls. The hits from FLAG-tagged twenty-four (TYF) proteomics were also excluded from the list to increase TANGO10-specificity. Proteins listed are those present in at least one elav-GAL4 sample and at least one tim-GAL4 sample but no negative controls.

2021-11-02 | PXD029326 | Pride

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing

Project description:ELAV/Hu factors are conserved RNA binding proteins that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, still little is known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, we find that ectopic expression of the three family members (Elav, Fne and Rbp9) induces overlapping changes to hundreds of cassette exon and dozens of alternative last exon (ALE) splicing events. Reciprocally, double mutants of elav/fne, but not elav alone, have opposite effects on both classes of regulated mRNA processing events in the larval CNS. While manipulation of Drosophila ELAV/Hu factors induces both exon skipping and inclusion, motif analysis indicates their major direct effects are to suppress cassette exon usage. Moreover, the roles of ELAV/HU factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both involve local suppression of proximal polyadenylation signals via ELAV/Hu binding sites downstream of cleavage sites. The phenotypic impact of combined ELAV/Hu activities in neural mRNA processing is overt, since fne loss strongly enhances neuronal differentiation phenotypes in elav mutants. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu proteins orchestrate multiple conserved programs of neuronal mRNA processing by suppressing alternative exons and polyadenylation sites.

2021-02-23 | GSE164129 | GEO

Transcription profiling of Drosophila adults to assess the effect of LARK overexpression in CNS neurons

Project description:The goal of this study is to identify, in the head of adult flies, mRNA species whose expresson level are altered by overexpression of the Drosophila RNA-binding protein LARK in CNS neurons. Experiment Overall Design: RNA samples from adult head of the LARK overexpression flies (elav-gal4; uas-lark/+) and control flies were compared. One total RNA sample was isolated from each genotype, of which three technical replicates (repeating the labeling and hybridization processes) were generated, respectively.

2008-06-14 | E-GEOD-6420 | biostudies-arrayexpress

Overlapping activities of ELAV/Hu family RNA binding proteins specify the extended neuronal 3' UTR landscape in Drosophila [S2 3-seq]

Project description:The goals of this study are to establish a cell line based analysis platform to unbiasedly evaluate the capabilities of Elav/Hu family RNA binding proteins in regulating neural specific 3' UTR extension; and perform quantitative analysis of global 3' UTR extension regulated by individual Elav/Hu family RNA binding protein. We reported that in non-neural tissue context such as S2R+ cells, with the overexpression of wildtype Elav, Rbp9 and Fne, we observed global 3' UTR extensions for hundreds of genes, whose 3' UTR extension corespond with those detected in head; also, RNA rocognition motif mutant versions of Elav, Rbp9 and Fne loss the ability to regulate the generation of neural specific 3' UTR extension. We reported here for the first time, we show quantitative comparison between Elav/Hu family RNA binding proteins in regulating neural specific 3' UTR landscape and imply the overlapping activities of Elav/Hu family RNA binding proteins specifying neural 3' UTR landscape in Drosophila.

2020-10-01 | GSE155533 | GEO

DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex

Project description:Alternative cleavage and polyadenylation (APA) is a form of transcriptional regulation via shifts in how frequently multiple polyadenylation (polyA) sites within genes are used across biological conditions. APA can result in transcripts with varying length and information content, and has been linked to gene regulation in both cancer and development. While trans-acting regulatory factors can cause changes in APA, the process of cleavage and polyadenylation is often co-transcriptional. We hypothesized that epigenetic changes, such as DNA methylation, may regulate APA co-transcriptionally. To identify genes where DNA methylation may regulate APA, we performed a genome-wide quantification of polyA site usage using a next generation sequencing technique. By priming the polyA-tails of an RNA library, we mapped cleavage sites and abundances with base-pair resolution across the genomes of two cell lines. The HCT116 cell line is derived from colon cancer, and contains cancer-associated DNA hypermethylation. The DKO cell line is derived from the parental HCT116 line, but has a near-complete depletion of DNA methylation due to mutations in DNA methyltransferase enzymes. Previous studies have used this sytem to detect genes where promoter DNA methylation regulates the gene expression of tumor suppressors. Analogously, detecting APA between HCT116 and DKO reveals candidate genes where DNA methylation may regulate the process of APA. Mechanistic experiments can further interrogate cross-talk between DNA methylation and APA at these loci, and contribute to the understanding of the function of non-promoter differentially methylated regions.

2020-03-18 | GSE86178 | GEO

Genome-wide profiling of Alternative polyadenylation sites in mouse female germline stem cells

Project description:Alternative polyadenylation (APA) is an important post-transcriptional modification implicated in development. Female germline stem cell (FGSC) is unipotent and capable of giving rise to oocyte. However, whether alternative polyadenylation plays a role in self-renew and cell fate determination of FGSCs remain elusive. Here, we used 3T-Seq developed in our lab to profile genome-wide 3a termini of transcripts and delineate APA sites in mouse FGSCs and explored the biological significance of APA modulation in FGSC identity.

2015-11-20 | E-MTAB-4038 | biostudies-arrayexpress

Molecular Organization of Drosophila Neuroendocrine Cells by DIMMED: Global Profiling of Pan-Neuronal DIMMED Expression Effects

Project description:To amass candidate DIMM targets in addition to Phm (Park et al., 2008a), we used genome-wide microarray profiling by over-expressing DIMM throughout the embryonic nervous system. We compared profiles from experimental (elav>dimm) and control (elav-GAL4) embryos at 22-26 hr and 28-32 hr after egg laying (AEL). The design was intended to identify transcripts consistently up-regulated shortly after the induction of DIMM; in so doing, we could circumvent the lethality that ensues in late embryonic, and/ or by early larval stages, due to pan-neuronal DIMM expression. Eight microarrays were used to hybridize cDNAs from experimental dimm::myc-expressing and control flies, sampled at two different embryonic stages. Four microarrays were hybridized with cDNA from the dimm::myc-expressing embryos of genotype w;UAS-dimm2A3/+;elav-GAL4/+; (prefix 'ED'). Another four microarrays were hybridized with cDNAs from the control (maternal) strain: w;;elav-GAL4 (prefix 'E'). Samples ED3 and ED4, E3 and E4 were hybridized with cDNA from stage 14 embryos (24-26hr development at 18C). Samples ED1 and ED2, E1 and E2 were hybridized with cDNA from stage 16 embryos (28-32 hr old embryos at 18C). A pair of GeneChip Drosophila Genome 2.0 arrays (Affymetrix Co., Santa Clara, CA) were tested with each RNA sample (two experimental and two control, for each of two time points). Affymetrix GeneChip Drosophila Genome 2.0 Array

2011-09-05 | E-GEOD-31113 | biostudies-arrayexpress

Alternative cleavage and polyadenylation analysis in an isoform-resolved way by Second- and Third-Generation Sequencing technologies

Project description:Alternative cleavage and polyadenylation (APA) is emerging as an important mechanism of gene regulation in eukaryotes and plays important regulatory roles in human development and diseases. Despite the widespread application of Second Generation Sequencing (SGS) technology for polyadenylation site identification, matching each identified polyadenylation site within a gene to its derived isoform remains a major challenge. To achieve the isoform-resolved APA analysis, we developed a tool termed “IDP-APA” that constructs truly expressed isoforms and identifies polyadenylation sites by integrating the respective strengths of Third Generation Sequencing (TGS) long reads and SGS short reads. Compared to existing tools, IDP-APA demonstrated superior performance in both isoform reconstruction and polyadenylation site identification. Applications to human embryonic stem cells, breast cancer cells and brain tissue from a patient with Alzheimer’s disease revealed prevalent APA events and cell-/tissue-specific APA patterns, especially in an isoform-resolved way.

2017-06-01 | GSE97688 | GEO

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