To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3′ end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3′ end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.
The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8α polypeptide chains and heterodimers of CD8α- and CD8β-chains. The function of the CD8α-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8αα homodimers. CD8αβ functions as a better coreceptor, but the actual function of CD8β is less clear. Addressing this issue has been hampered by the apparent inability of CD8β to be expressed without CD8α. This study demonstrates that human, but not mouse, CD8β can be expressed on the cell surface without CD8α in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8β is responsible for the lack of expression of murine CD8ββ dimers. In contrast to CD8αα, CD8ββ is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 β-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8ββ Ig domain. In addition, we demonstrate that the Ig domains of CD8α are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8α reduced the ability of the protein to fold properly and, therefore, to be expressed.
In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.
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