A novel Cd8-cis-regulatory element preferentially directs expression in CD44hiCD62L+ CD8+ T cells and in CD8αα+ dendritic cells

Abstract

CD8 coreceptor expression is dynamically regulated during thymocyte development and is tightly controlled by the activity of at least 5 different cis-regulatory elements. Despite the detailed characterization of the Cd8 loci, the regulation of the complex expression pattern of CD8 cannot be fully explained by the activity of the known Cd8 enhancers. In this study, we revisited the Cd8ab gene complex with bioinformatics and transgenic reporter gene expression approaches to search for additional Cd8 cis-regulatory elements. This led to the identification of an ECR (ECR-4), which in transgenic reporter gene expression assays, directed expression preferentially in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like CD8(+) T cells. ECR-4, designated as Cd8 enhancer E8VI, was bound by Runx/CBFβ complexes and Bcl11b, indicating that E8VI is part of the cis-regulatory network that recruits transcription factors to the Cd8ab gene complex in CD8(+) T cells. Transgenic reporter expression was maintained in LCMV-specific CD8(+) T cells upon infection, although short-term, in vitro activation led to a down-regulation of E8VI activity. Finally, E8VI directed transgene expression also in CD8αα(+) DCs but not in CD8αα-expressing IELs. Taken together, we have identified a novel Cd8 enhancer that directs expression in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like and antigen-specific effector/memory CD8(+) T cells and in CD8αα(+) DCs, and thus, our data provide further insight into the cis-regulatory networks that control CD8 expression.

Heritable Gene Regulation in the CD4:CD8 T Cell Lineage Choice

“…In transgenic studies, E8 V (CII) exhibited no enhancer function by itself, but a combination of E8 V and E8 I directed expression in both CD8 + T cells and DP thymocytes, unlike E8 I alone, suggesting that stage-specific activity could arise through combinations of CD8 enhancers (107). Recently, another cis element termed E8 VI (CIV-6) was described, which directed transgenic reporter expression in mature CD8 + T cells, memory-phenotype-like CD44 hi CD62L + CD8 + T cells, and CD8αα + dendritic cells (108). In summary, at least six enhancers direct CD8 expression in cytotoxic T cells (Figure 5).…”

Editorial: Crossing the divide: a novel Cd8 enhancer with activity in CTLs and CD8αα+ dendritic cells

“…The E8 I enhancer was not reported to have activity in DCs, another lineage that selectively expresses CD8a, indicating that additional regulatory elements remained to be identified. In the report by Sakaguchi et al [5], E8 VI transgene activity in CD8aa + DCs but not in CD8aa + IELs documents that E8 VI contributes to a novel CD8a regulatory pathway that does not depend on Runx3 or Bcl11b. In CTLs, both Runx3 and Bcl11b bind to E8 VI , consistent with the idea that in this cell type, E8 VI reinforces the activity of other Cd8 enhancers.…”

“…In the study by Sakaguchi et al [5] the focus returned to the primary DNA sequence of the Cd8 locus to identify novel CRE. The authors performed a cross-species comparison among the Cd8 locus of human, dog, rat, and mouse and identified 10 ECRs, where 6 of these regions overlapped with the previously studied E8 I , E8 II , and E8 V .…”

“…This bias of E8 VI activity to peripheral CTLs is unique, as previous reporters showed preference for thymic developmental subsets (E8 II , E8 III , and E8 IV ) or were found in thymic and peripheral compartments (E8 I ). Further characterization showed that reporter [5], indicating the relative location of Cd8b1 and Cd8a genes and the CRE contained in the Cd8 locus (E8 IV , E8 III , E8 II , E8 VI , E8 I , and E8 V ). The enhancer identified in the current study, E8 VI , is indicated in red.…”

“…The study by Sakaguchi et al [5] also evaluated reporter expression on non-CTL lineages from E8 VI transgenic mice.…”