Sophia D. Sarafova

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

ABSTRACT Expression of the CD4 gene is tightly controlled throughout thymopoiesis. The downregulation of CD4 gene expression in CD4− CD8− and CD4−CD8+ T lymphocytes is controlled by a transcriptional silencer located in the first intron of the CD4 locus. Here, we determine that the c-Myb transcription factor binds to a functional site in the CD4 silencer. As c-Myb is also required for CD4 promoter function, these data indicate that depending on the context, c-Myb plays both positive and negative roles in the control of CD4 gene expression. Interestingly, a second CD4 silencer-binding factor, HES-1, binds to c-Myb in vivo and induces it to become a transcriptional repressor. We propose that the recruitment of HES-1 and c-Myb to the silencer leads to the formation of a multifactor complex that induces silencer function and repression of CD4 gene expression.

A Potential Role for Elf-1 in CD4 Promoter Function

The control of CD4 gene expression is believed to be linked directly to the signaling events that mediate T cell development and is directly dependent on the CD4promoter. We have previously determined that this promoter contains four factor-binding sites important for its function. One of these sites, referred to as the P4 site, contains an Ets consensus recognition sequence. Using functional and biochemical analyses, we determine that Elf-1 binds to this site and specifically activates theCD4 promoter, indicating that Elf-1 is playing an important role in CD4 promoter function. In addition, a second nuclear factor binds to this region. Although there are consensus recognition sites for other factors, we demonstrate that none of these factors binds to the P4 site, nor do other known members of the Ets family. Thus, a novel transcription factor may bind to theCD4 promoter and help mediate its function.

Coreceptor Signal Strength Regulates Positive Selection but Does Not Determine CD4/CD8 Lineage Choice in a Physiologic In Vivo Model

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8α gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8α gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.

Upregulation of CD4 expression during MHC class II-specific positive selection is essential for error-free lineage choice.

The lineage fate of developing thymocytes is determined by the persistence or cessation of T cell receptor (TCR) signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We show here that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during major histocompatibility complex class II (MHC II)-specific positive selection and is critical for error-free lineage choice in TCR-transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurred for endogenously encoded CD4 coreceptors, but CD4 transgenes were downregulated during positive selection, disrupting MHC II-specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC II-specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice.

Targeting CD4 Coreceptor Expression to Postselection Thymocytes Reveals That CD4/CD8 Lineage Choice Is neither Error-Prone nor Stochastic

The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that CD4/CD8 lineage choice in TCR-signaled CD4+CD8+ thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by “coreceptor rescue” experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8I-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8I-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8I-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8I-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic.

Control of CD4 gene expression: connecting signals to outcomes in T cell development

The control of CD4 gene expression is essential for proper T lymphocyte development. Signals transmitted from the T-cell antigen receptor (TCR) during the thymic selection processes are believed to be linked to the regulation of CD4 gene expression during specific stages of T cell development. Thus, a study of the factors that control CD4 gene expression may lead to further insight into the molecular mechanisms that drive thymic selection. In this review, we discuss the work conducted to date to identify and characterize the cis-acting transcriptional control elements in the CD4 locus and the DNA-binding factors that mediate their function. From these studies, it is becoming clear that the molecular mechanisms controlling CD4 gene expression are very complex and differ at each stage of development. Thus, the control of CD4 expression is subject to many different influences as the thymocyte develops.