Promoter Knock-In Mutations Reveal a Role of Mcl-1 in Thymocyte-Positive Selection and Tissue or Cell Lineage-Specific Regulation of Mcl-1 Expression

Abstract

We previously demonstrated that IL-3 stimulates transcription of the antiapoptotic gene mcl-1 via two promoter elements designated as the SIE and CRE-2 sites. To further study the functional role of these two DNA elements, mutant mice with targeted mutations of both SIE and CRE-2 sites (SC mutants) were generated. Homozygous SC mutants manifested a markedly reduced level of Mcl-1 in thymus but not in other major organs such as spleen, liver, lung, or heart. Reduced expression of Mcl-1 in SC mutant thymus resulted in attenuated positive selection of double-positive thymocytes into both CD4 and CD8 lineages, a result likely due to reduced survival of SC mutant double-positive thymocytes that were supposed to be positively selected. In contrast, in the peripheral lymphoid organs, only CD8؉ but not CD4 ؉ T cells were significantly reduced in homozygous SC mutant mice, a result consistent with a more dramatic decrease both of Mcl-1 expression and cell viability in mutant CD8؉ compared with mutant CD4؉ T cells. Impaired T cell development and peripheral CD8 ؉ lymphopenia in homozygous SC mutant mice were both cell autonomous and could be rescued by enforced expression of human Mcl-1. Together, the promoter-knock-in mouse model generated in this study not only revealed a Mcl-1, a member of the Bcl-2 family protein, was originally identified in the ML-1 human myeloid leukemia cell line that underwent phorbol ester-induced differentiation (1). Mcl-1 is widely expressed in various tissues and cell lineages, and a great number of studies indicate that Mcl-1 plays an apical role in many cell survival and death pathways (2). Mcl-1 deficiency results in embryonic lethality at the peri-implantation stage (3). Conditional knock-out approaches have thus been used to assess Mcl-1 functions in other tissues. Using this approach, Mcl-1 has been shown to be essential for the survival of many cell types in the hematopoietic system, including hematopoietic stem cells, lymphoid precursors, mature T and B lymphocytes, and neutrophils (4 -6).T cell progenitors arise from the bone marrow and migrate into the thymus to become early committed T cells that lack expression of TCR, CD4, and CD8. These cells are termed double-negative (DN) 3 thymocytes (7). DN thymocytes then develop in an ordered progression and mature into the CD4 ϩ CD8 ϩ double-positive (DP) stage (8). At this stage, immature DP thymocytes expressing unique TCRs are tested individually by two selection processes that eliminate cells whose TCRs either cannot recognize self peptide/MHC complex (positive selection) or react too strongly (negative selection). These two selection processes ensure that most DP thymocytes selected to differentiate into CD4 single-positive (SP) or CD8SP thymocytes, and eventually peripheral T cells, can respond properly when encountering foreign peptides in the context of self MHC in the periphery (8 -10). Many Bcl-2 family members have been shown to play a role in T cell development. Deletion of Bcl-2 resulted in rapid loss of thymocytes and naive...

Expression of cell cycle and apoptosis regulators in thymus and thymic epithelial tumors

“…For instance, (a) the expression levels of Bcl-2, Mcl-1, Bim and Bax were higher in medullary than in cortical thymocytes, whereas Bcl-xL exhibited the opposite expression pattern, (b) the expression levels of Bax, Bak, Bad and Mcl-1 were higher in medullary than in cortical TEC and Bcl-2 was expressed by some medullary TEC and (c) Bid was frequently expressed by thymocytes and medullary TEC [32]. These findings are consistent with flow cytometry and Western blot results in thymocytes [96][97][98][99][100][101][102] and provide evidence that the Bcl-2 family members are differentially involved in the cell survival and death events during the differentiation of TEC and thymocytes.…”

Critical Roles of Translationally Controlled Tumor Protein in the Homeostasis and TCR-Mediated Proliferation of Peripheral T Cells

“…After 3 days, lymph nodes and spleens from the recipient mice were analyzed for the presence of CFSE-labeled cells. This assay gave a similar result to that obtained by injecting T cells into a congenic strain of mice, and following the presence of injected T cells using Abs specific for congenic markers (19).…”

“…In vivo cell survival assay was conducted as previously described (19). Briefly, Mature SP thymocytes (CD4 or CD8) were purified from control or Lck-TCTP Ϫ/Ϫ mice and labeled with CFSE (Molecular Probes) for 10 min at 37°C.…”

Elevated Mcl-1 inhibits thymocyte apoptosis and alters thymic selection

“…22 Furthermore, mice with reduced thymic Mcl-1 expression had defective positive selection in the H-Y model. 34 Taken together, these data suggest that endogenous Mcl-1 levels are limiting for positive selection and important for CD8SP maturation.…”