Identification of proteins interacting with lncRNA Braveheart in mouse using protein microarray
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Zhihong Xue
PROVIDER: E-MTAB-4995 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Transcription factors (TFs) play a central role in regulating gene expression by interacting with cis regulatory DNA elements associated with their target genes. Recent surveys have examined the DNA binding specificities of most Saccharomyces cerevisiae transcription factors but a comprehensive evaluation of their data has been lacking. Results: We analyzed in vitro and in vivo TF-DNA binding data reported in previous large-scale studies to generate a comprehensive, curated resource of DNA binding specificity data for all characterized S. cerevisiae transcription factors. Our collection comprises DNA binding site motifs and comprehensive in vitro DNA binding specificity data for all possible 8 bp sequences. Included in this database is DNA binding specificity data for 27 TFs independently generated by PBM analysis in this current study. Investigation of the DNA binding specificities within the basic leucine zipper (bZIP) and VHR transcription factor families revealed unexpected plasticity in TF-DNA recognition: intriguingly, the VHR transcription factors, newly characterized by protein binding microarrays in this study, recognize bZIP like DNA motifs, while the bZIP transcription factor Hac1 recognizes a motif highly similar to the canonical E-box motif of basic helix-loop-helix (bHLH) transcription factors. We identified several transcription factors with distinct primary and secondary motifs, which might be associated with different regulatory functions. Finally, integrated analysis of in vivo transcription factor binding data with protein binding microarray data lends further support for indirect DNA binding in vivo by sequence-specific transcription factors. 27 Protein binding microarray (PBM) experiments of Saccharomyces cerevisiae transcription factors were performed. Briefly, the PBMs involved binding GST-tagged yeast transcription factors to double-stranded 44K Agilent microarrays in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473). A key feature is that the microarrays are composed of de Bruijn sequences that contain each 10-base sequence once and only once, providing an evenly balanced sequence distribution. Individual de Bruijn sequences have different properties, including representation of gapped patterns. The array probe sequences on the custom array design used in this study were reported previously in Berger et al., Cell 2008 (PMID 18585359) and are available via an academic research use license. Here we provide the data transformed into median signal intensities (after normalization and detrending of the original array data) for all 32,896 8-base sequences, Z-scores for these intensities, and E-scores. E-scores are a modified version of AUC and describe how well each 8-mer ranks the intensities of the spots. In general, the E-scores are slightly more reproducible than Z-scores, but contain less information about relative binding affinity. Additional experimental details are found in Berger et al., Nature Biotechnology 2006, Gordan et al., Genome Biology (in press), and the accompanying Supplementary information.
2011-12-10 | E-GEOD-34306 | biostudies-arrayexpress
Project description:Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SUUR protein is found at late-replicating intercalary heterochromatin sites and in pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in the non-polytenic Drosophila Kc cells by using DamID. This approach is based on the ability of a chromatin protein fused to Escherichia coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled (using Cy3 or Cy5) and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000). We generated both an N- and a C-terminal fusion of the full-length SuUR open reading frame with Dam (Dam-SUUR and SUUR-Dam, respectively). For each SUUR fusion protein we performed four independent replicates. We used for this study a cDNA array developed by the GeneCore facility in EMBL (Heidelberg, Germany), covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome. We found that SUUR preferentially binds to genes that are transcriptionally silent and late replicated. We compared the SUUR binding profile to the binding profile of three PcG proteins, which are known to bind to many intercalary heterochromatin sites, and found that there is a significant overlap with Pc and esc, but less with Sce. A significant overlap is also detected with two markers of pericentric heterochromatin, the heterochromatin proteins HP1 and SU(VAR)3-9. Finally, we demonstrated that SUUR binding profile negatively correlates with DNA polytenization level in salivary gland polytene chromosomes. Taken together, these results suggest that SUUR modulates the level of underreplication by direct binding to intercalary and pericentric heterochromatin.
2007-07-04 | E-MEXP-863 | biostudies-arrayexpress
Project description:Although 3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been predominately linked to PI3K-AKT pathway, it may also evoke additional signaling outputs to promote tumorigenesis. Here we report that PDK1 directly induces phosphorylation of Polo-like kinase 1 (PLK1), which in turn induces Myc phosphorylation and protein accumulation. We show that PDK1-PLK1-Myc signaling is critical for cancer cell growth and survival and small molecule inhibition of PDK1/PLK1 provides an effective approach for therapeutic targeting Myc-dependency. Intriguingly, PDK1-PLK1-Myc signaling induces an embryonic stem cell-like gene signature associated with aggressive tumor behaviors and is a robust signaling axis driving cancer stem cell (CSC) self renewal. Finally, we show that PLK1 inhibitor synergizes with mTOR inhibitor to induce synergistic anti-tumor effect in colorectal cancer by antagonizing a compensatory Myc induction. These findings identify a novel pathway in human cancer and CSC activation and provide a therapeutic strategy for targeting Myc-associated tumorigenesis and therapeutic resistance. Gene expression profiling of Human Embryonic Kidney Cells (HEK-TERV) under different conditions: PMN, PDK1, MYC and E545K
2013-07-31 | E-GEOD-30669 | biostudies-arrayexpress
Project description:HeLa cell extracts with or without GSK3 enzyme inhibition were assayed using protein microarrays in order to detect GSK3-dependent changes in protein polyubiquitination. HeLa lysates in triplicates were supplemented with ubiquitin and incubated on protein microarrays (ProtoArray 5.0; Invitrogen) in the presence or absence of the GSK3 inhibitor SB-216763. Polyubiquitination of the arrayed proteins was detected using specific antibodies. ProtoArray 5.0 contains over 9,000 full-length human proteins purified and arrayed in duplicate under native conditions to maximize functionality.
2014-05-15 | E-GEOD-50629 | biostudies-arrayexpress
Project description:The transcription factor E2F4 is a member of the E2F family of regulators which has critical roles in cell cycle control and differentiation. We investigated the genome-wide binding profile of E2F4 using a newly developed peak calling program based on a kernel density estimation and identified approximately 16 thousands E2F4 binding sites at a 1% FDR threshold, which potentially modulate 7,346 downstream target genes. Gene Ontology (GO) functional analysis of E2F4 target genes revealed several novel functions of E2F4 including I-kappaB kinase/NF-kappaB cascade, protein transport and targeting, protein folding, and sterol biosynthetic process. In addition to mRNA target genes, E2F4 also binds and regulates some miRNA targets such as let-7a, mir-17 and mir-22. Interestingly, we found that about 20% of E2F4 sites are in intergenic regions, which suggests that E2F4 may function at enhancer sites. De novo motif discovery revealed 5 novel, significantly enriched motifs within E2F4 sites. Only 5% of binding sites contain a canonical motif, indicating that E2F4 can use diverse motifs to bind DNA and regulate transcription. Overexpression of E2F4 and its cofactors such as RBL2 and DP-1, followed by microarray analysis, revealed that E2F4 functions as an activator and a repressor. Taken together, our genome-wide E2F4 ChIP sequencing data provided evidences of versatile physiological roles of E2F4 and insights into its functions. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2010-11-01 | E-GEOD-21489 | biostudies-arrayexpress
Project description:Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem-cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila Kc cells. We found that Pc associates with large genomic regions of up to ~150kb in size, hereafter referred to as âPc-domainsâ. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 lysine 27 and in general transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains. Keywords: DamID, Chromatin immunoprecipitation, ChIP-chip To study PcG binding profiles we used DamID, which is based on the ability of a chromatin protein fused to E.coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000).
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2022-03-07 | PXD031606 | Pride
Project description:About half of all human and mouse miRNA genes are located within introns of protein-coding genes. Despite this, little is known about functional interactions between miRNAs and their host genes. The intronic miRNA miR-128 regulates neuronal excitability and controls dendrite outgrowth of projection neurons during development of the mouse cerebral cortex. Its host genes R3hdm1 and Arpp21 encode highly conserved, putative RNA-binding proteins. Here we use iCLIP to describe the RNA-binding activity of ARPP21, which recognizes uridine-rich sequences with exquisite sensitivity for 3UTRs. Surprisingly, ARPP21 antagonizes miR-128 activity by co-regulating a subset of miR-128 target mRNAs enriched for neurodevelopmental functions. In contrast to miR-128, we show that ARPP21 acts as a positive post-transcriptional regulator, at least in part through interaction with the eukaryotic translation initiation complex eIF4F. This molecular antagonism is also reflected in inverse activities during dendritogenesis: miR-128 overexpression or knockdown of ARPP21 reduces dendritic complexity; ectopic ARPP21 leads to an increase. The regulatory interaction between ARPP21 and miR-128 is a unique example of convergent function by two products of a single gene.
2018-02-23 | E-MTAB-5911 | biostudies-arrayexpress
Project description:In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 34% of the fly genome is organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and share biological functions. Furthermore, fewer chromosomal rearrangements occur inside chromatin domains than outside domains during evolution. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution. Keywords: DamID DamID experiments for multiple chromatin proteins was performed in Drosophila cell cultures. Samples were hybridized to spotted cDNA arrays. Every experiment was repeated at least 2 times, with one sample in the reverse dye orientation. When hybridization was performed 4 times, two samples were hybridized in the reverse dye orientation.
2008-04-07 | E-GEOD-10219 | biostudies-arrayexpress