Negative Regulation of CD4 Gene Expression by a HES-1–c-Myb Complex

T-cell development is controlled by the ordered regulation of genes involved in the progression of the thymocyte through each stage of maturation. One of the most important genes expressed at specific stages of T-cell development is that encoding the coreceptor CD4. The earliest committed T-cell precursor cells do not express either CD4 or the coreceptor CD8 and are referred to as double-negative (DN) thymocytes. Expression of CD4 and CD8 is first seen in T cells that have undergone successful rearrangement of the T-cell receptor (TCR) β genes. This double-positive (DP) population subsequently completes rearrangement of the TCR α chain gene and undergoes the selection process to ensure a properly restricted T-cell repertoire (6). CD4 expression is maintained in mature T cells that survive selection and recognize antigen bound to major histocompatibility complex class II (5, 15). These cells downregulate expression of CD8 and become committed helper T cells (TH). Those cells that survive selection and recognize antigen bound to major histocompatibility complex class I will downregulate expression of CD4, maintain expression of CD8, and become committed cytotoxic T cells (TC) (42, 46). Thus, the activation or downregulation of CD4 gene expression defines the different stages of developing T cells. We have sought to understand how CD4 gene expression is linked to thymocyte development by identifying factors that bind to and mediate the function of the CD4 transcriptional control regions. As these trans-acting factors are likely to be responsive to the T-cell selection process, their study will help delineate the signaling pathways that drive repertoire selection. Expression of the CD4 gene is controlled by four elements: a promoter, a thymocyte enhancer which functions early in development, a mature enhancer which begins function in mature post-selection T cells, and a transcriptional silencer (1, 8–10, 33, 34, 39, 40, 42, 43, 47, 49, and M. Adlam and G. Siu, unpublished). The silencer is the critical element that represses CD4 expression in DN thymocytes and as the DP thymocyte matures into the CD8 single-positive (SP) TC cell (41, 43). There are three factor-binding sites in the CD4 silencer, which we refer to as S1, S2, and S3, all of which are important in mediating silencer function (10). We have previously determined that the Notch pathway intermediate HES-1 binds to the silencer site S1 and silencer-associated factor (SAF) binds to S3; both are important in mediating silencer function (22, 23). Interestingly, SAF is located in the cytoplasm of DP and CD4 SP T cells and in the nucleus of DN and CD8 SP T cells. Its active transport to the nucleus is DP thymocyte specific, is controlled by lck signaling via the Mek1 pathway, and induces the downregulation of CD4 expression, the initiating step of CD8 SP development (W. W. S. Kim, N. de Souza, and G. Siu, submitted for publication). It is thus likely that SAF is critical for transmitting signals from the TCR complex to the CD4 silencer during the repertoire selection process. The factor(s) that binds to the S2 site of the silencer is unknown. The c-Myb transcription factor plays a role in the control of transcription of many genes that are critical for early hematopoiesis (25); mouse genetic studies have also demonstrated a role for c-Myb in early thymopoiesis (2, 4). Consensus c-Myb-binding sites have been found in the transcriptional control elements of genes important in later stages of thymopoiesis, indicating that c-Myb may also play a role in late T-cell development as well (12, 17, 19, 28, 44). We and others have previously determined that c-Myb induces CD4 expression by binding to a consensus c-Myb site in the CD4 promoter (28, 44). Here, using molecular and transgenic approaches, we determine that c-Myb binds to the S2 functional site of the CD4 silencer and that its binding is important for mediating silencer function. In addition, we demonstrate that HES-1 binds to c-Myb in vivo and induces it to become a transcriptional repressor. Our data thus indicate that c-Myb can function as either a transcriptional repressor at the CD4 silencer or as a transcriptional activator at the CD4 promoter, depending on the context of its binding site within the transcriptional control element, and indicate that HES-1 and c-Myb form a multifactor complex that mediates CD4 silencer function.