Abstract
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.
Highlights
Gene expression is coordinated by an interplay among different regulatory regions, which work together to determine precisely when, where, and how they are transcribed
In this study we targeted the endogenous CNS2 element by CRISPR-Cas9 deletion in various human cell lines and primary T cells to evaluate its function in a native chromatin context
The CNS2 regulatory region is necessary for transcription initiation and promotion, but it displays orientation-independent activity in driving and potentiating transcriptional output, functions traditionally attributed to enhancer elements
Summary
Gene expression is coordinated by an interplay among different regulatory regions, which work together to determine precisely when, where, and how they are transcribed. The orchestration of gene transcriptional programs relies on two main players: promoters and distal regulatory elements. Whereas promoters are located immediately upstream of gene transcriptional start sites (TSS), distal regulatory elements act over long genomic distances. Basal expression from promoters is modulated by a combination of signals from distal regulatory elements, which determine the final transcriptional output. Enhancers and promoters have been considered to be two different regulatory elements on the basis of chromatin characteristics, such as DNase hypersensitivity and chemical modification of histone tail residues. Current trends support a unique regulatory feature with varying degrees of overlap of promoter and enhancer potential (Andersson and Sandelin, 2020)
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