A Promoter in the Coding Region of the Calcium Channel Gene CACNA1C Generates the Transcription Factor CCAT
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{"title"=>"A Promoter in the Coding Region of the Calcium Channel Gene CACNA1C Generates the Transcription Factor CCAT", "type"=>"journal", "authors"=>[{"first_name"=>"Natalia", "last_name"=>"Gomez-Ospina", "scopus_author_id"=>"6507094288"}, {"first_name"=>"Georgia", "last_name"=>"Panagiotakos", "scopus_author_id"=>"9733774800"}, {"first_name"=>"Thomas", "last_name"=>"Portmann", "scopus_author_id"=>"7801656199"}, {"first_name"=>"Sergiu P.", "last_name"=>"Pasca", "scopus_author_id"=>"12792452800"}, {"first_name"=>"Dania", "last_name"=>"Rabah", "scopus_author_id"=>"7801386516"}, {"first_name"=>"Agata", "last_name"=>"Budzillo", "scopus_author_id"=>"55651408000"}, {"first_name"=>"Jean Pierre", "last_name"=>"Kinet", "scopus_author_id"=>"7101746200"}, {"first_name"=>"Ricardo E.", "last_name"=>"Dolmetsch", "scopus_author_id"=>"35563932800"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84876167239", "sgr"=>"84876167239", "pui"=>"368726767", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"23613729", "doi"=>"10.1371/journal.pone.0060526"}, "id"=>"eec84175-ffe9-35a4-b65d-21c2a8d0d032", "abstract"=>"The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3' end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.", "link"=>"http://www.mendeley.com/research/promoter-coding-region-calcium-channel-gene-cacna1c-generates-transcription-factor-ccat", "reader_count"=>34, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>10, "Student > Ph. D. Student"=>7, "Student > Master"=>4, "Other"=>1, "Student > Bachelor"=>5, "Professor"=>5}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>10, "Student > Ph. D. Student"=>7, "Student > Master"=>4, "Other"=>1, "Student > Bachelor"=>5, "Professor"=>5}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>8, "Agricultural and Biological Sciences"=>13, "Medicine and Dentistry"=>4, "Neuroscience"=>6, "Philosophy"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Neuroscience"=>{"Neuroscience"=>6}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>8}, "Unspecified"=>{"Unspecified"=>2}, "Philosophy"=>{"Philosophy"=>1}}, "reader_count_by_country"=>{"United States"=>4, "Portugal"=>2}, "group_count"=>4}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1026107"], "description"=>"<p>(A) Immunohistochemistry of E18, P1, and 3-week-old rat cortex, cerebellum, and thalamus showing developmental variation in the amount and distribution of CCAT nuclear staining. Cortex shows mostly cell body and dendritic staining of CCAT in the three developmental stages. Anti-CCAT is shown in red and nuclei in blue. (B) Immunohistochemistry of E18 mouse sagittal sections through the cortex, ventricle, and thalamus stained with anti-CCAT antibody. (C) Immunocytochemistry of cortical and thalamic neurons grown 5 days <i>in vitro</i> stained with anti-CCAT. Transcriptional assays of cortical (top) and thalamic (bottom) neuronal cultures transfected with the UAS-luciferase reporter along with the Gal4-tagged channels described in Fig. 1. Bars represent normalized transcription to Gal4 alone. (Means ± SD; * <0.005 and ** <0.0001 vs. M2011I-Gal4).</p>", "links"=>[], "tags"=>["Computational biology", "Molecular genetics", "gene expression", "developmental biology", "stem cells", "Induced pluripotent stem cells", "genetics", "DNA transcription", "genomics", "Genome complexity", "neuroscience", "Cellular neuroscience", "ion channels", "regulated"], "article_id"=>683728, "categories"=>["Biological Sciences"], "users"=>["Natalia Gomez-Ospina", "Georgia Panagiotakos", "Thomas Portmann", "Sergiu P. Pasca", "Dania Rabah", "Agata Budzillo", "Jean Pierre Kinet", "Ricardo E. Dolmetsch"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060526.g004", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CCAT_Expression_is_Regulated_During_Brain_Development_/683728", "title"=>"CCAT Expression is Regulated During Brain Development.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-16 01:02:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/1026108"], "description"=>"<p>(A) Schematic representation of <i>CACNA1C</i> showing the location of TSS's found and depictions of the proteins predicted to be expressed from these transcripts. (B) Table showing a summary of transcriptional start sites and nearby CAGE tags. Chromosomal addresses for experimental TSS are given as the corresponding location in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding). (C) HEK cells expressing Mem-CCAT GFP and CCAT GFP constructs. (D-E) Western blot analysis of membrane fractions (D) and nuclear fractions (E) obtained from 11.5 dpc heterozygous (N/+) or homozygous (N/N) Ca<sub>v</sub>1.2 knockout embryos probed with the anti-CCAT antibody. Bottom panels show loading controls. (F) Immunohistochemistry of 11.5 dpc Ca<sub>v</sub>1.2 null embryos reveals strong nuclear staining with the anti-CCAT antibody (red) in the developing somites. Nuclei are shown in blue.</p>", "links"=>[], "tags"=>["Computational biology", "Molecular genetics", "gene expression", "developmental biology", "stem cells", "Induced pluripotent stem cells", "genetics", "DNA transcription", "genomics", "Genome complexity", "neuroscience", "Cellular neuroscience", "ion channels", "sites"], "article_id"=>683729, "categories"=>["Biological Sciences"], "users"=>["Natalia Gomez-Ospina", "Georgia Panagiotakos", "Thomas Portmann", "Sergiu P. Pasca", "Dania Rabah", "Agata Budzillo", "Jean Pierre Kinet", "Ricardo E. Dolmetsch"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060526.g005", "stats"=>{"downloads"=>2, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transcriptional_Start_Sites_in_the_3_End_of_CACNA1C_Produce_Multiple_Proteins_/683729", "title"=>"Transcriptional Start Sites in the 3′ End of <i>CACNA1C</i> Produce Multiple Proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-16 01:02:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1026110", "https://ndownloader.figshare.com/files/1026112", "https://ndownloader.figshare.com/files/1026114", "https://ndownloader.figshare.com/files/1026115", "https://ndownloader.figshare.com/files/1026117"], "description"=>"<div><p>The C-terminus of the voltage-gated calcium channel Ca<sub>v</sub>1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Ca<sub>v</sub>1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of <i>CACNA1C,</i> the gene that encodes Ca<sub>V</sub>1.2. Expression of CCAT is independent of Ca<sub>v</sub>1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of Ca<sub>V</sub>1.2. Analysis of the transcriptional start sites in <i>CACNA1C</i> and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3′ end of the <i>CACNA1C</i> gene. This study provides new insights into the regulation of <i>CACNA1C,</i> and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.</p></div>", "links"=>[], "tags"=>["Computational biology", "Molecular genetics", "gene expression", "developmental biology", "stem cells", "Induced pluripotent stem cells", "genetics", "DNA transcription", "genomics", "Genome complexity", "neuroscience", "Cellular neuroscience", "ion channels", "promoter", "coding", "calcium", "generates", "transcription", "ccat"], "article_id"=>683731, "categories"=>["Biological Sciences"], "users"=>["Natalia Gomez-Ospina", "Georgia Panagiotakos", "Thomas Portmann", "Sergiu P. Pasca", "Dania Rabah", "Agata Budzillo", "Jean Pierre Kinet", "Ricardo E. Dolmetsch"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0060526.s001", "https://dx.doi.org/10.1371/journal.pone.0060526.s002", "https://dx.doi.org/10.1371/journal.pone.0060526.s003", "https://dx.doi.org/10.1371/journal.pone.0060526.s004", "https://dx.doi.org/10.1371/journal.pone.0060526.s005"], "stats"=>{"downloads"=>8, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Promoter_in_the_Coding_Region_of_the_Calcium_Channel_Gene_CACNA1C_Generates_the_Transcription_Factor_CCAT/683731", "title"=>"A Promoter in the Coding Region of the Calcium Channel Gene <i>CACNA1C</i> Generates the Transcription Factor CCAT", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-04-16 01:02:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1026101"], "description"=>"<p>(A) Schematic representation of the Ca<sub>v</sub>1.2-Gal4 fusion and channel mutants. Four mutations are depicted: ΔTM-IQ is a deletion from the TM to IQ motif that renders the channel unable to traffic to the membrane. ΔC.S is a deletion of the conserved cleavage site in Ca<sub>v</sub>1.1. STOP is a translational stop at 1910 AA. TA is a deletion of CCAT's transcription activation domain. (B) Western blot of Neuro2A cells expressing Ca<sub>v</sub>1.2-Gal4 channels depicted in A (upper panel) and Gal4-tagged C-terminal fragments (bottom panel) probed with an antibody to Gal4. (C) Reporter gene activity of Neuro2A cells expressing a UAS-luciferase reporter plasmid along with Ca<sub>v</sub>1.2-Gal4 channels depicted in A or Gal4 alone as a control. Cells were co-transfected with a Renilla luciferase construct driven by the thymidine kinase promoter to control for cell number and transfection efficiency. Results are given as a ratio of Firefly to Renilla luciferase activity. (Means ± SD; *<0.0001 vs. Gal4). (D) Western blot of Neuro2A cells expressing WT, M2011I, and M2078I Ca<sub>v</sub>1.2-Gal4 channels (Upper Panel). Bottom panel shows Gal4-tagged C-terminal fragments. Proteins were detected with a Gal4 antibody. Large molecular protein in channel western represents unsolubilized, multimeric channel proteins. (E) Luciferase activity of Neuro2A cells expressing either Gal4 alone, WT, M2011I, or M2078I Gal4-tagged channels. (Means ± SD; * <0.0001 vs. Gal4). (F) Mean luciferase activity (± SD) in Neuro2A cells expressing CCAT-Gal4 constructs along with the UAS-luciferase reporter and TK-Renilla luciferase construct as controls. CCAT-ΔTA lacks the transcriptional activation domain and serves as a negative control. CCAT-IFIL corresponds to WT sequence. Sequences are included to show the mutations used.</p>", "links"=>[], "tags"=>["Computational biology", "Molecular genetics", "gene expression", "developmental biology", "stem cells", "Induced pluripotent stem cells", "genetics", "DNA transcription", "genomics", "Genome complexity", "neuroscience", "Cellular neuroscience", "ion channels"], "article_id"=>683723, "categories"=>["Biological Sciences"], "users"=>["Natalia Gomez-Ospina", "Georgia Panagiotakos", "Thomas Portmann", "Sergiu P. Pasca", "Dania Rabah", "Agata Budzillo", "Jean Pierre Kinet", "Ricardo E. Dolmetsch"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060526.g001", "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CCAT_Expression_is_Independent_of_Ca_v_1_2_Channel_Protein_/683723", "title"=>"CCAT Expression is Independent of Ca<sub>v</sub>1.2 Channel Protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-16 01:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1026103"], "description"=>"<p>(A) Northern blot analysis of mRNA extracted from Neuro2A cells expressing Ca<sub>v</sub>1.2-Gal4 channel constructs with or without CMV promoter. The first lane contains mRNA extracted from untransfected cells. The membranes were hybridized with a radioactively labeled RNA probe to Exon 47 of the channel. (B) Western blot of Neuro2A cells expressing Ca<sub>v</sub>1.2-Gal4 channel constructs with or without CMV promoter. Upper panel shows full-length channels. Bottom panels shows Gal4-tagged CCAT. Membranes were immunoblotted with a Gal4 antibody. (C) UAS reporter activity of Neuro2A cells expressing Ca<sub>v</sub>1.2-Gal4 channel constructs with or without CMV promoter. (Means ± SD; *<0.0001 vs. Gal4). (D) Schematic representation of Firefly reporter constructs designed to map the region within the coding sequence of the channel responsible for the promoter activity. Full-length construct has the complete sequence of the channel upstream of the luciferase coding sequence. E1-45 construct includes all exons up to exon 45. E46-47 contains exon 46 and 47 upstream of luciferase. (E) Luciferase activity as a surrogate measure of luciferase expression from constructs depicted in D. Graph compares expression level in Neuro2A cells transfected with either full-length, E1-45, E46-47, E46, or E47 constructs. (Means ± SD). (F) Schematic representation of the minigenes. A 4 Kb genomic segment containing the last two exons and introns of <i>CACNA1C</i> was fused to the coding sequence of Gal4. WT is the wild-type minigene. FS is a negative control where a G has been inserted between exon 47 and Gal4. Δ238bp lacks the 238bp region identified in E. Met 2011 has been mutated to isoleucine in M2011. (G) Mean luciferase activity (± SD) in Neuro2A cells expressing WT, FS, Δ238bp, and M2011I Gal4 minigenes along with a UAS-luciferase reporter. (* <0.0001 vs. WT).</p>", "links"=>[], "tags"=>["Computational biology", "Molecular genetics", "gene expression", "developmental biology", "stem cells", "Induced pluripotent stem cells", "genetics", "DNA transcription", "genomics", "Genome complexity", "neuroscience", "Cellular neuroscience", "ion channels", "translated", "transcript", "driven", "exonic"], "article_id"=>683724, "categories"=>["Biological Sciences"], "users"=>["Natalia Gomez-Ospina", "Georgia Panagiotakos", "Thomas Portmann", "Sergiu P. Pasca", "Dania Rabah", "Agata Budzillo", "Jean Pierre Kinet", "Ricardo E. Dolmetsch"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060526.g002", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CCAT_is_Translated_from_an_Independent_Transcript_Driven_by_an_Exonic_Promoter_/683724", "title"=>"CCAT is Translated from an Independent Transcript Driven by an Exonic Promoter.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-16 01:02:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1026105"], "description"=>"<p>(A–B) Northern blot analysis of mRNA extracted from cortex, diencephalon, and cerebellum from E18, P1, and adult rats. The membranes were hybridized with radioactively labeled DNA probe to either exon 47 (A) or exons 21–24 (B) of the channel. Bottom panel shows the same membrane labeled with a probe to the 18S ribosomal RNA as a loading control. (C) Graphs showing the normalized expression of the full-length and 2.2 Kb band in the cortex, midbrain, and cerebellum at 3 developmental stages: E18, P1, and adult. Each line represents an independent experiment. The northern blot shown in A corresponds to the blue tracing. Signals were normalized to the 18S RNA signal. (D–E) Dot plots showing the normalized expression of exons 2, 8, 44, and 47 per cell in mouse neurons. For all comparisons of exon 47 to the other exons, p<0.001 (***) according to one-way ANOVA with post-hoc Bonferroni correction for multiple comparisons. Exons 2, 8, and 44 are not significantly different from each other. (F) Graphs showing fractions of human IPSC-derived neurons expressing exon 47 exclusively or both exons 47 and 8 (n = 269). (G) Fraction of exon 47-containing human IPSC-derived neurons expressing either exon 47 exclusively or co-expressing exon 8.</p>", "links"=>[], "tags"=>["Computational biology", "Molecular genetics", "gene expression", "developmental biology", "stem cells", "Induced pluripotent stem cells", "genetics", "DNA transcription", "genomics", "Genome complexity", "neuroscience", "Cellular neuroscience", "ion channels", "generated", "transcript"], "article_id"=>683726, "categories"=>["Biological Sciences"], "users"=>["Natalia Gomez-Ospina", "Georgia Panagiotakos", "Thomas Portmann", "Sergiu P. Pasca", "Dania Rabah", "Agata Budzillo", "Jean Pierre Kinet", "Ricardo E. Dolmetsch"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0060526.g003", "stats"=>{"downloads"=>5, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CCAT_is_Generated_From_an_Independent_Transcript_in_vivo_/683726", "title"=>"CCAT is Generated From an Independent Transcript <i>in vivo</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-16 01:02:06"}

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Relative Metric

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