Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression
The CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed...
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doaj-01121cb889ed4b1e874d099000e729dc2020-11-25T03:56:35ZengFrontiers Media S.A.Frontiers in Genetics1664-80212020-10-011110.3389/fgene.2020.552949552949Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 ExpressionJennifer Redondo-Antón0MG Fontela1Laura Notario2Raúl Torres-Ruiz3Sandra Rodríguez-Perales4Elena Lorente5Pilar Lauzurica6Immune Gene Regulation and Antigen Presentation Group, National Center for Microbiology, Institute of Health Carlos III (ISCIII), Madrid, SpainImmune Gene Regulation and Antigen Presentation Group, National Center for Microbiology, Institute of Health Carlos III (ISCIII), Madrid, SpainImmune Gene Regulation and Antigen Presentation Group, National Center for Microbiology, Institute of Health Carlos III (ISCIII), Madrid, SpainMolecular Cytogenetics and Genome Editing Unit, Spanish National Cancer Research Centre (CNIO), Madrid, SpainMolecular Cytogenetics and Genome Editing Unit, Spanish National Cancer Research Centre (CNIO), Madrid, SpainImmune Gene Regulation and Antigen Presentation Group, National Center for Microbiology, Institute of Health Carlos III (ISCIII), Madrid, SpainImmune Gene Regulation and Antigen Presentation Group, National Center for Microbiology, Institute of Health Carlos III (ISCIII), Madrid, SpainThe CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed to the participation of complex gene regulatory networks. An evolutionarily conserved regulatory element, CNS2, located 4kb upstream of the CD69 gene transcriptional start site, has been proposed as the major candidate governing the gene transcriptional activation program. To investigate the function of human CNS2, we studied the effect of its endogenous elimination via CRISPR-Cas9 on CD69 protein and mRNA expression levels in various immune cell lines. Even when the entire promoter region was maintained, CNS2-/- cells did not express CD69, thus indicating that CNS2 has promoter-like characteristics. However, like enhancers, inverted CNS2 sustained transcription, although at a diminished levels, thereby suggesting that it has dual promoter and enhancer functions. Episomal luciferase assays further suggested that both functions are combined within the CNS2 regulatory element. In addition, CNS2 directs its own bidirectional transcription into two different enhancer-derived RNAs molecules (eRNAs) which are transcribed from two independent transcriptional start sites in opposite directions. This eRNA transcription is dependent on only the enhancer sequence itself, because in the absence of the CD69 promoter, sufficient RNA polymerase II levels are maintained at CNS2 to drive eRNA expression. Here, we describe a regulatory element with overlapping promoter and enhancer functions, which is essential for CD69 gene transcriptional regulation.https://www.frontiersin.org/articles/10.3389/fgene.2020.552949/fullCD69immune regulationenhancerpromoterenhancer-derived RNA (eRNA)transcriptional regulation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jennifer Redondo-Antón MG Fontela Laura Notario Raúl Torres-Ruiz Sandra Rodríguez-Perales Elena Lorente Pilar Lauzurica |
spellingShingle |
Jennifer Redondo-Antón MG Fontela Laura Notario Raúl Torres-Ruiz Sandra Rodríguez-Perales Elena Lorente Pilar Lauzurica Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression Frontiers in Genetics CD69 immune regulation enhancer promoter enhancer-derived RNA (eRNA) transcriptional regulation |
author_facet |
Jennifer Redondo-Antón MG Fontela Laura Notario Raúl Torres-Ruiz Sandra Rodríguez-Perales Elena Lorente Pilar Lauzurica |
author_sort |
Jennifer Redondo-Antón |
title |
Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression |
title_short |
Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression |
title_full |
Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression |
title_fullStr |
Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression |
title_full_unstemmed |
Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression |
title_sort |
functional characterization of a dual enhancer/promoter regulatory element leading human cd69 expression |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Genetics |
issn |
1664-8021 |
publishDate |
2020-10-01 |
description |
The CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed to the participation of complex gene regulatory networks. An evolutionarily conserved regulatory element, CNS2, located 4kb upstream of the CD69 gene transcriptional start site, has been proposed as the major candidate governing the gene transcriptional activation program. To investigate the function of human CNS2, we studied the effect of its endogenous elimination via CRISPR-Cas9 on CD69 protein and mRNA expression levels in various immune cell lines. Even when the entire promoter region was maintained, CNS2-/- cells did not express CD69, thus indicating that CNS2 has promoter-like characteristics. However, like enhancers, inverted CNS2 sustained transcription, although at a diminished levels, thereby suggesting that it has dual promoter and enhancer functions. Episomal luciferase assays further suggested that both functions are combined within the CNS2 regulatory element. In addition, CNS2 directs its own bidirectional transcription into two different enhancer-derived RNAs molecules (eRNAs) which are transcribed from two independent transcriptional start sites in opposite directions. This eRNA transcription is dependent on only the enhancer sequence itself, because in the absence of the CD69 promoter, sufficient RNA polymerase II levels are maintained at CNS2 to drive eRNA expression. Here, we describe a regulatory element with overlapping promoter and enhancer functions, which is essential for CD69 gene transcriptional regulation. |
topic |
CD69 immune regulation enhancer promoter enhancer-derived RNA (eRNA) transcriptional regulation |
url |
https://www.frontiersin.org/articles/10.3389/fgene.2020.552949/full |
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