Notch3 and the Notch3-upregulated RNA-binding protein HuD regulate Ikaros alternative splicing

Abstract

Constitutive activation of the transmembrane receptor, Notch3, and loss of function of the hematopoietic transcription repressor, Ikaros (IK), play direct roles in T‐cell differentiation and leukemogenesis that are dependent on pre‐T‐cell receptor (pre‐TCR) signaling. We demonstrate the occurrence of crosstalk between Notch3 and IK that results in transcriptional regulation of the gene encoding the pTα chain of the pre‐TCR. We also show that, in the presence of the pre‐TCR, constitutive activation of Notch3 in thymocytes causes increased expression of dominantnegative non‐DNA‐binding IK isoforms, which are able to restrain the IK inhibition of Notch3's transcriptional activation of pTα. This effect appears to be mediated by Notch3's pre‐TCR‐dependent upregulation of the RNA‐binding protein, HuD. Notch3 signaling thus appears to play a critical role in the diminished IK activity described in several lymphoid leukemias. By exerting transcription‐activating and transcription‐repressing effects on the pTα promoter, Notch3 and IK cooperate in the fine‐tuning of pre‐TCR expression and function, which has important implications for the regulation of thymocyte differentiation and proliferation.

Acetylation controls Notch3 stability and function in T-cell leukemia

“…In agreement with our present observations and with previous data demonstrating that Notch signaling activation in pre-T cells or in bone marrow results in the accumulation of CD4 þ CD8 þ DP cells in spleen and lymph nodes (Pear et al, 1996;Beverly et al, 2005;Bellavia et al, 2007), this feature being a characteristic of T-cell leukemia, we observed a decreased Notch3 protein expression specifically in splenic CD4 þ CD8 þ DP cells of TSA-treated mice. Therefore, it may be speculated that, by impairing N3 IC signaling, HDACi block the expansion or migration of CD4 þ CD8 þ cells, possibly representing the precursors of leukemic cells, in peripheral lymphoid organs and in circulating blood.…”

“…TSA-induced downregulation of Notch3 prevents T-ALL development and progression in N3 IC tg mice The role of Notch3 acetylation in enhancing protein degradation would be consistent with the subsequent suppression of Notch3-dependent transcriptional activity and activation of a number of proliferative or oncogenic pathways (Talora et al, 2003(Talora et al, , 2006Vacca et al, 2006;Bellavia et al, 2007). Overall, these findings suggest that this acetylation mechanism might be exploited for controlling Notch3-dependent leukemia.…”

“…Therefore, the impaired ability of acetylated Notch3 to enhance pTa expression implies its control of T-cell differentiation and tumorigenesis. Indeed, T-cell differentiation, whose mis-regulation results in leukemogenesis, has been previously reported to be disrupted by aberrant activation of Notch3 in N3 IC trangenic mice, by transcriptionally activating the pTa gene (Bellavia et al, 2007). The resulting constitutive activation of pre-TCR signaling in tg N3 IC mice is indispensable in T-cell lymphomagenesis, as the loss of the pTa gene in N3 IC / pTa-null double mutant mice prevents T-cell leukemia development (Bellavia et al, 2002).…”

“…We and others previously showed that accumulation of CD4 þ CD8 þ DP cells in spleen, lymph nodes and peripheral blood represents a pathognomonic feature of T-cell leukemias sustained by enforced expression of N IC in pre-T-cells or in bone marrow of mice (Pear et al, 1996;Bellavia et al, 2000Bellavia et al, , 2007. Figures 6e and 7a, respectively, show that the cell population most importantly affected by the TSA treatments is represented by the CD4 þ CD8 þ DP T cells in both spleen and lymph nodes when compared with vehicle-injected littermates, either when the percent distribution or the absolute number is considered.…”

Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia

“…c-Cbl activity in Notch3-dependent leukemia S Checquolo et al Notch3 and pTa association was also detected in 2017 pre-T-cell line (Spolski et al, 1988) that express high level of both Notch3 and pTa (Bellavia et al, 2007) (Figure 4e, right panel). Altogether these observations suggest that the interaction of Notch3 and pTa proteins is possible at the cell surface of thymocytes, where the lipid rafts represent the molecular platform in which TCR or pre-TCR components cluster to trigger intracellular signaling.…”