- Leukemogenesis, uncontrolled proliferation of dysfunctional, transformed immature lymphocytes, appears, especially in children, to be associated with abrogation of normal lymphopoiesis, and thymopoiesis in the case of T cell leukemias. Sequence specific transcription factors (SSTFs) are nuclear control switches that respond to cellular signals to alter cell state and cell fate by controlling gene transcription. Their activities are highly and precisely regulated by diverse means such as alterations in protein stability, post-translational modifications (PTMs) and/or protein-protein interactions. Several SSTFs have been identified as “T cell lineage-determining factors” essential for proper maturation of thymocytes into T cells in immune system development. Mutations or dysregulation of these T cell lineage-specifying SSTFs are associated with leukemogenesis.
SSTFs act in coordination to regulate gene transcription in multi-protein complexes. There are millions of potential combinations of SSTFs available to fine-tune repression or activation of individual genes in response to multiple, complex extracellular and intracellular signaling inputs. To narrow our studies to SSTFs most likely to be of consequence in leukemogenesis, we focused on modifications and interactions between lineage-determining SSTFs in the thymocyte maturation process. Tcf1 is a T cell lineage-determining SSTF and has been identified as the nuclear effector of Wnt/Gsk3/β-catenin -dependent signaling. Bcl11b is a nuclear target of the T-cell receptor (TCR)-mediated MAP Kinase (MAPK) signaling cascade, but there is no indication from the literature that an interaction between Tcf1 and Bcl11b has been investigated. As an impetus for this investigation, another group demonstrated the ability of Bcl11b to regulate Wnt target genes in a different system, but a molecular link between the Wnt signaling pathway and Bcl11b was not established. Through our studies in primary murine thymocytes (mostly double positive (DP) cells), we identified a previously unknown physical interaction between Bcl11b and Tcf1, and among Bcl11B, TCF1 and β-catenin in Jurkat cells, a human T cell leukemia cell line. Our further studies in mutant Jurkat cells revealed that neither sumoylation nor up to 285 C terminal amino acids in Bcl11b are required for BCL11B to complex with TCF1 or β-catenin. We identified several thousand gene promoter regions that are co-occupied by the two factors, suggesting that the Bcl11b-Tcf1 interaction may coordinately regulate many genes required for thymocyte maturation. Using a gene reporter assay, we identified that the Bcl2l1 gene, encoding for the anti-apoptotic protein Bcl-xl, is co-regulated by BCL11B and TCF1.
Treatment of thymocytes to mimic stimulation of the Wnt/Gsk3 signaling pathway also resulted in significant changes in the PTMs of Bcl11b, including increased sumoylation and decreased phosphorylation. Previous studies identified two kinases that are involved in modification of Bcl11b, Erk1/2 and p38 MAPK; in this study we identified a third candidate kinase, Gsk3, that may be responsible for basal phosphorylation. Inhibition of Gsk3 in thymocytes, mimicking canonical Wnt pathway activation, reduced the Tcf1-Bcl11b interaction, suggesting a potential mechanism by which Bcl11b regulates Wnt target genes.
By investigating DN3-like P2C2 cells, a cell line from an earlier stage in thymocyte development relative to DP cells, we observed different patterns of PTM kinetics in response to signaling pathway activation, as compared to primary thymocytes. Treatment to mimic pre-TCR/MAP kinase pathway activation in P2C2 cells mostly replicated findings in primary thymocytes in terms of BCL11B PTM kinetic changes, with the P2C2 cells having a slightly delayed time course overall. However, this delay creates a situation in which desumoylation and dephosphorylation occurs simultaneously, in contrast to the opposing modifications by sumoylation and phosphorylation observed in DP thymocytes in response to MAPK pathway activation. Nevertheless, the opposite regulation of phosphorylation and sumoylation that we observed in primary thymocytes is mimicked under other conditions in the DN-like cells. Hyper-phosphorylation of Bcl11b after treatment with a broad phosphatase inhibitor coincided with near complete desumoylation. Additionally, activation of Wnt/Gsk3-dependent signaling in P2C2 cells resulted in composite dephosphorylation and sumoylation of Bcl11b. P2C2 cells displayed a higher basal level of β-catenin than primary thymocytes, and a higher ratio of long (β-catenin responsive) to short (β-catenin independent) Tcf1 isoforms. Unlike in DP thymocytes, we did not observe an interaction between Bcl11b and Tcf1 in the DN-like cells. Our results demonstrate that PTMs and specific interacting partners of Bcl11b in P2C2 cells are distinct from those previously characterized in DP thymocytes.
Our results contribute to the existing knowledge regarding dynamic regulation of Bcl11b PTMs, and the interaction of Bcl11b with specific transcriptional complexes during distinct stages of T cell development. These findings may contribute to a better understanding of the molecular mechanisms that regulate Bcl11b transcriptional activity in different cell types, potentially leading to novel treatments for T cell leukemias.