A genome-wide analysis of open chromatin in human epididymis epithelial cells reveals novel candidate regulatory elements for male infertility
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Ann Harris
PROVIDER: E-GEOD-51003 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were over-represented in epididymis-selective DHS identified epithelial transcription factors including ELF5 and ELF3, the androgen receptor, Pax2 and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq.
2013-09-19 | E-GEOD-51002 | biostudies-arrayexpress
Project description:The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were over-represented in epididymis-selective DHS identified epithelial transcription factors including ELF5 and ELF3, the androgen receptor, Pax2 and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium. HEE cells were cultured as described previously (Harris and Coleman 1989). RNA was isolated by Trizol extraction from three primary cultures of HEE cells. Total RNA was purified by Millipore Microcon YM-100 filter centrifugation and shipped to MoGene, MO, for gene expression analysis on Nimblegen 4 x 72K HG18 60mer arrays. This submission represents transcriptome component of study.
2013-09-06 | E-GEOD-50605 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-11-08 | E-GEOD-52181 | biostudies-arrayexpress
Project description:Distal cell-type-specific regulatory elements may be located at very large distances from the genes that they control and are often hidden within intergenic regions or in introns of other genes. The development of methods that enable mapping of regions of open chromatin genome wide has greatly advanced the identification and characterisation of these elements. Here we use DNase I hypersensitivity mapping followed by deep sequencing (DNase-seq) to generate a map of open chromatin in primary human tracheal epithelial (HTE) cells and use bioinformatic approaches to characterise the distribution of these sites within the genome and with respect to gene promoters, intronic and intergenic regions. Genes with THE-selective open chromatin at their promoters were associated with multiple pathways of epithelial function and differentiation. The data predict novel cell-type-specific regulatory elements for genes involved in HTE cell function, such as structural proteins and ion channels, and the transcription factors that may interact with them to control gene expression. Moreover, the map of open chromatin can identify the location of potentially critical regulatory elements in genome-wide association studies (GWAS) in which the strongest association is with single nucleotide polymorphisms in non-coding regions of the genome. We demonstrate its relevance to a recent GWAS that identifies modifiers of cystic fibrosis lung disease severity. Since HTE cells have many functional similarities with bronchial epithelial cells and other differentiated cells in the respiratory epithelium, these data are of direct relevance to elucidating the molecular basis of normal lung function and lung disease. To identify cis-regulatory elements for genes expressed in human tracheal epithelial cells we generated genome-wide maps of open chromatin by DNase-seq. HTE cells were isolated from these trachea and grown as described previously (Davis P.B. et al.1990).
2013-11-08 | E-GEOD-52179 | biostudies-arrayexpress
Project description:Investigation of gene expression level changes in snapdragon petals and sepals during flower development The flower developmental stages analyzed in this study are representative of distinct developmental events: (i) preanthesis, (ii) anthesis, (iii) maturation and (iv) presenescence and are further described in the accompanying article. A 24 chip study using total RNA recovered from samples of petal and sepal tissue of Antirrhinum majus cv. Maryland True Pink harvested at four different stages of flower development, namely (i) preanthesis (three days before flower opening=d-3), (ii) anthesis (day of flower opening=d1), (iii) maturation (four days after flower opening=d4) and (iv) presenescence (seven days after flower opening=d7). Three separate samples were extracted per tissue and developmental stages. Each chip measures the expression level of 11,959 ESTs from Antirrhinum majus cv. Maryland True Pink with up to six 60-mer probes per target.
2012-06-06 | E-GEOD-36356 | biostudies-arrayexpress
Project description:A transgenic line cmlc2:TRAP was made to express EGFP-fused ribosomal protein L10a (EGFP-L10a) in zebrafish cardiomyocytes. Then ribosome-associated RNAs were immuoprecipitated from uninjured and injured adult cmlc2:TRAP fish to determine the differential expression changes during zebrafish heart regeneration. A nine chip study with cardiomyocyte ribosome associated RNAs purified from three separate isolation of uninjured adult cmlc2:TRAP fish hearts, three separate isolation of 1 day post amupation (dpa) adult cmlc2:TRAP fish hearts, and three separate isolation of 7 dpa adult cmlc2:TRAP fish hearts.
2013-08-01 | E-GEOD-48914 | biostudies-arrayexpress
Project description:Bryophytes are the most basal of the extant land plants. A major feature of these plants is the biphasic alteration of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. To analyse the differences in the transcriptome of the early gametophyte (protonema) and early and mid-sporophyte phases of the moss Physcomitrella patens, microarray gene expression profiles were performed using dissected sporophyte tissue. Through further analysis the early and mid-sporophyte phases were compared. RNA isolated from the Gametophytic protonemal tissue was hybridised to six microarrays. Each microarray was hybridised with RNA from a separate biological replicate. Three of these microarrays were co-hybridised with RNA isolated from early sporophytes. With the third gametophyte biological replicate and early sporophyte replicate a dye swap was carried out. The remaining three microarrays hybridised with RNA from the gametophytes were co-hybridised with RNA from mid-sporophytic tissue. A dye swap was carried out on the sixth gametophyte replicate and third mid-sporophyte replicate.To meet the quality requirements for the microarray experiment, at least 400 sporophytes were used per extraction. Three or four RNA extracts were then pooled for further precipitation to maximise purity and concentration. Up to 1600 sporophytes were harvested to prepare sufficient RNA for each microarray replicate. In bioinformatic analysis the channels were split into individual channels and the early and mid-sporophyte were compared.
2013-07-29 | E-GEOD-32928 | biostudies-arrayexpress
Project description:Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the phosphatidylinositol 3-kinase (PI3K) related kinase family that contain a C- terminal PI3K domain followed by a ~ 35-residue FATC domain. We have characterized four alleles with single residue changes in the FATC domain. Of these tra1-L3733A had the most pronounced effects with phenotypes including temperature and cold sensitivity, and reduced growth in media containing ethanol, Calcofluor white, rapamycin, chloramphenicol and geneticin. Tra1-L3733A interacted at normal levels with components of the NuA4 and SAGA complexes, and did not significantly alter histone acetylation patterns. The tra1-L3733A allele resulted in two-fold or greater change in expression of approximately 11% of yeast genes in rich media. Of the 279 genes with increased expression, 175 were ribosomal subunits or involved in ribosomal function or biogenesis. Elevated levels of Pol I and Pol III transcripts were also observed. The phenotypes of the tra1-L3733A overlapped with but were not identical to strains containing deletions of SAGA or NuA4 components or with strains containing mutations in the PI3K domain. Our finding that the double mutant allele, tra1-SRR3413/L3733A with alterations in the PI3K and FATC domains, resulted in wild type growth, suggests a model whereby the FATC domain negatively regulates the activity of the PI3K domain. Expression of genes involved in ribosome biosynthesis, other than the ribosomal subunits themselves, returned to near normal levels in the double mutant strain. We also characterized tra1-G3745, which contains an additional glycine residue following the normal C-terminal phenylalanine. This allele did not support viability and showed severe dominant negative effects. In contrast to what was observed for tra1-L3733A, tra1-G4745 resulted in decreased expression of genes required for ribosome biogenesis and did not interact with Esa1 or Spt7. Three biological replicate experiments including one dye-swap were performed for yeast strains CY3003(TRA1::Tn10LUK with IB150(myc9-tra1_L3733A-YCplac111)) and CY3015(TRA1::Tn10LUK with IB157(myc9-tra1_SRR3413_L3733A-YCplac111)) with reference to CY2706(TRA1::Tn10LUK with 1980(myc9-TRA1-YCplac111)). Similarly, three biological replicate experiments including one dye-swap were performed for yeast strain CY3019(TRA1::Tn10LUK with 1259(myc-TRA1-YCplac111) and IB162(myc9-tra1_G3745-YCplac111)) with reference to CY3020(TRA1::Tn10LUK with 1259(myc-TRA1-YCplac111) and IB160(myc9-TRA1-YCplac111)).
2013-01-30 | E-GEOD-13561 | biostudies-arrayexpress
Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.
2012-12-19 | E-GEOD-42981 | biostudies-arrayexpress
Project description:In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123. The SAGA complex component, Ubp8, is one of two proteases that remove this ubiquitin moiety. We analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affects gene expression. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1K123R. This provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Second, microarray analyses of the htb1K123R strain show that loss of histone ubiquitylation results in a two-fold or greater change in expression of ~1.5% of the protein coding genes with greater than two-thirds increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of the methyltransferase components Swd3, Set2 or Dot1 and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1K123R strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of its being a site of ubiquitylation. Keywords: yeast, histone ubiquitylation, Ubp8, gene expression, genetic modification, histone H2B Two dye-swapped, biological replicate experiments were performed for yeast strains CY1272(Htb1_K123R;htb2_delta0), BY10809(ubp8_delta0) and CY1383(Htb1_K123R;htb2_delta0;ubp8_delta0) with reference to BY4742(wt). Three biological replicates, including one dye-swap experiment, were performed comparing CY1272(Htb1_K123R;htb2_delta0) to BY13026(htb2_delta0).
2010-09-15 | E-GEOD-6316 | biostudies-arrayexpress