Molecular analysis of CD8 gene expression

Cell fate specifications during thymocyte differentiation are linked with the tightly controlled expression of CD4 and CD8 coreceptor molecules. Therefore, understanding the regulation of Cd4 and Cd8 gene expression might not only provide insight into transcriptional control mechanisms during T cell development, but may also help to explain the molecular basis of lineage commitment and function. The expression of the adjacent genes Cd8a and Cd8b is controlled by at least five developmental stage-, subset- and lineage-specific cis-regulatory elements, and we could link Cd8 enhancer function with chromatin remodeling of the Cd8 gene loci. Despite this detailed characterization of the Cd8ab gene complex, several important issues remain to be addressed. It is not known whether Cd8 enhancers are required at all stages during T cell development or whether CD8 expression can be epigenetically maintained in the absence of Cd8 enhancers. It is also not known whether the same or different CD8 enhancers are involved in the regulation of CD8 upon T cell activation. Moreover, the Cd8 cis-regulatory elements that are required to direct expression of CD8a in DCs have not been identified, and it remains to be determined whether negative-acting cis-regulatory elements (Cd8 "silencer") or repressive transcription factors are required to shut-off CD8 expression during CD4 T cell lineage differentiation. In this joint project proposal, we propose to address some of these questions with mouse molecular genetics tools (Cre/loxP mediated gene targeting strategies) and we will revisit the Cd8ab gene complex with bioinformatics approaches to identify novel Cd8 cis-regulatory elements. Furthermore, we propose to test the role of ThPOK and Runx complex in the regulation of Cd8 gene expression.

T cells for a subgroup of white blood cells and have important functions in the control of immune responses. They can be subdivided into two major subsets, the so-called CD4+ helper T cells and the CD8+ cytotoxic T cells. Both T cells subsets develop in the thymus from a so-called CD4 and CD8 double-positive thymocytes. We are interested to understand how this CD4 (i.e. helper) versus CD8 (i.e. cytotoxic) cell fate decision of double-positive (DP) thymocytes is regulated. During T cell development, the expression of CD4 and CD8 coreceptors is dynamically regulated and tightly linked to the generation of helper and cytotoxic T cells. Thus, understanding co-receptor gene expression will provide insight into CD4/CD8 cell fate choice. During T cell development, CD8 coreceptor expression is regulated by the activity of at least five different cis-regulatory elements. We recently linked Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b (Cd8) and demonstrated epigenetic control of the Cd8 gene complex. Despite this detailed characterization of the Cd8ab gene complex, several important issues remain to be addressed. For instance, it was not known whether Cd8 enhancers are required at all stages during T cell development or whether CD8 expression can be epigenetically maintained in the absence of Cd8 enhancers. In addition, the Cd8 cis-regulatory elements that are required to direct expression of CD8? in DCs have not been identified. With the support of the joint project proposal, we revisited the Cd8ab gene complex with bioinformatics and mouse molecular genetics approaches to identify novel Cd8 cis-regulatory elements and to further characterize already known Cd8 enhancers. Our studies revealed a novel and unexpected role for the Cd8 enhancer E8I and Runx/CBF? complexes in the regulation and maintenance of Cd8a gene expression in CD8+ effector T cells, and indicate different mechanisms of how CD8 expression is regulated in nave and effector T cells.Moreover, we identified a novel Cd8 enhancer that directs expression in CD44hiCD62L+ CD8+ T cells including innate-like CD8+ T cells and in CD8??+ DCs. Taken together, the results from the FWF project I698 provide further insight into the cis-regulatory network that controls CD8 expression.