Studies here define the regions within the CD5 promoter that control transcriptional regulation in resting (unstimulated) T cells growing in culture. By constructing a deletion series of CD5 promoter constructs using luciferase as the reporter gene, we demonstrate that a 43 bp region between the StuI and HincII sites at -215 to -172 is necessary for normal, resting CD5 expression level in T cells. Further, we show that this region contains an Ets binding site that regulates CD5 transcription, two previously unrecognized regions that also control CD5 expression, one of which is responsible for controlling basal expression of the promoter.
Weichert and Schwartz [25
] have also reported a deletion analysis of the CD5 promoter. Several of their deletions overlap the deletions we report. For those constructs which were similar in both studies, our results agree. However, these workers observed negative regulation with a construct that that we did not have. None of our constructs showed negative regulation. We investigated three potential transcription factor binding sites (CCAAT, κE2, and Ets) within the 43 bp regulatory promoter region and showed that only the Ets binding site influences CD5 expression in EL4 T cells. Further, we showed that the Ets-1 protein binds to the Ets site and that overexpression of Ets-1 leads to increased promoter activity. Thus we conclude that Ets-1 positively regulates CD5 expression levels in T cells.
Theses findings are consistent with previous evidence showing that Ets-1 levels in T cell subsets correlate with the expression of CD5 on these subsets (see Introduction to this paper). In addition, they are consistent with evidence demonstrating that CD5 expression is drastically reduced in gene targeted mice in which the Ets-1 locus is disrupted. While the possibility that the decreased CD5 surface expression that was demonstrated in the gene targeted mice is due to post-translational rather than transcriptional effects, our co-transfection studies with the Ets-1 expression plasmid and the CD5 promoter constructs demonstrate argue that Ets-1 is regulating transcription in all cases. Thus, the weight of our findings, in combination with findings with presented by others, leads us to conclude that the distinctive CD5 expression levels observed in T cell subsets is due to the different amounts of Ets-1 protein in the cells in these subsets. This conclusion does not eliminate the possibility that Ets family members other than Ets-1 also participate in the regulation of CD5 expression. PU.1, for example, could be important in the regulation of CD5 expression on B-1 cells, since it is predominantly expressed in B cells and macrophages [35
]. In fact, preliminary evidence from our laboratory suggests that this may indeed be the case. Ets-2, which is ubiquitously expressed, could also contribute to CD5 regulation [29
]. However, the data from Ets-1 gene targeted mice argues against this possibility since disruption of the Ets-1 gene is sufficient to prevent CD5 expression on T cells. Therefore, Ets-1 is the likely regulatory molecule in T cells.
We also characterized two regions (CD5X and CD5Y) that contribute to CD5 expression. These two regions, which contain previously unrecognized transcription binding factor sequence, are conserved between mouse and human. Our results showed that the CD5Y region is necessary for basal level of CD5 transcription. Computer analysis of the CD5Y region did not reveal any potential transcription factor binding site or a canonical TATA box for establishing the typical RNA polymerase II transcription complex. However, the Ymut1 site contains a very weak homology to the TdT/ML intiator consensus sequence, 5'-PyPyCAPyPyPyPyPy-3' [39
]. The Ymut2 site, which is the most important site for controlling basal expression, overlaps a potential initiator site fitting the consensus (5' CTT CCC TTT TCC 3') for the ribosomal protein Inr family with only one mismatched base [40
]. Interestingly, both Ymut1 and Ymut2 are positioned near the most upstream transcriptional start site [25
The initiator (Inr) element has been shown to be critical in positioning RNA polymerase II complex for transcription initiation in TATA-less promoters [39
]. Since the CD5 promoter does not contain a TATA box, these initiator binding sites may play a more important role in establishing transcription initiation.
Several Inr binding protein have been identified: TFII-I [41
], YY1 [42
], and a 150 kDa TATA binding protein (TBP) associated factor (TAFII
]. Recognition of the Inr element by these Inr binding proteins (ITF) provides a way for formation of transcription competent complex via their interaction with TBP or with other subunits of RNA polymerase II. Thus we suggest that CD5Y region interacts with these ITFs to regulate transcription.
Like CD5Y, CD5X region does not contain any consensus transcription factor binding sites. This region is not necessary for basal CD5 transcription but it affects the overall level of CD5 transcription obtained with the full length 3 Kb promoter. At least three specific protein:DNA complexes are formed in EMSA using the CD5X oligonucleotides. We speculate that proteins binding to the CD5X region may interact with other upstream transcription factors, e.g., Ets-1, rather than with proteins binding to the CD5Y region. This interaction, we suggest, acts to stabilize a transcriptional competent complex and thereby to increase the CD5 expression to normal levels.
This suggestion underlies the model proposed in figure 6 for regulating CD5 expression in T cells. In this model, the CD5Y region sets up the basal transcription machinery by interacting with components in the RNA polymerase II transcription complex and/or with transcription initiators, since CD5 lacks a TATA box for establishing a typical RNA polymerase II trancription initiation complex. The CD5X region recruits transcription factors to the upstream 43 bp region identified as necessary for overall CD5 expression.
The proteins binding to the CD5X region in this model recruit Ets-1 protein (and perhaps other Ets family members) to the Ets binding site. Alternatively, these proteins help to alleviate the internal Ets-1 inhibition domains and stabilize the binding of Ets-1 to its cognitive site [33
]. In any event, they facilitate the interaction of Ets-1 with other factors to form activate transcription and increase it from the basal to the overall level.
Our studies here focus on characterizing the promoter elements responsible for regulating the constitutive CD5 expression in unstimulated T cells. These elements, we have shown, exist in a region proximal to the translation start and quite distant from an enhancer element shown in other studies [46
] to regulate the activation of CD5 expression in BCR-stimulated LPS-induced B cell blasts. This enhancer element, located at approximately 2 kb upstream of the translation start on the CD5 promoter, is dependent on NF-AT activation to induce CD5 expression in anti-IgM stimulated primary splenic cells cultured with LPS for 3 days [48
]. It is reported to interact with proteins in the proximal region of the CD5 promoter, which contains the all three regions characterized here.
An Ets binding site also has been implicated in the activity of the enhancer discussed above. In addition, this CD5 response to BCR stimulation has been shown to be mediated in part by vav-2, which activates of NF-AT [49
]. Our studies with constitutive expression of CD5 on B cells are still in progress. However, preliminary evidence suggests that unlike the stimulated CD5 expression in these other studies, the low-level constitutive expression of CD5 on the M12 B cell line is enabled by the same 43 bp regulatory region that we have shown here is sufficient to induce full constitutive CD5 expression on T cells.