Notch controls binary cell fate decisions during development and adult tissue renewal to produce and maintain normal tissue architecture. To achieve this effect, Notch signals work together with an intricate network of nuclear factors that collectively coordinate and integrate incoming signals to direct complex phenotypes. Central to the effects of Notch on cell fate specification is the conversion of CSL from being a repressor to an activator of gene expression in response to Notch-activating stimuli. Before Notch activation, CSL nucleates assembly of transcriptional repressor complexes on Notch-responsive promoters. Several proteins have been implicated in CSL-mediated transcriptional repression, including NCoR/SMRT (26
), SHARP/MINT/SPEN (41
), CIR (21
), SKIP (54
), HAIRLESS (39
), and CtBP and Groucho/TLE (8
). MTG family proteins expand the roster of corepressors that bind CSL and reinforce the notion that stringent repression of gene expression is desired to control Notch-dependent phenotypes. Recently, Salat et al. showed that MTG8 enhances repression by SHARP at RBP-Jκ
responsive promoters and that knockdown of MTG8 increases Notch target gene expression without overt Notch receptor activation (44
). Similarly, we found enhanced expression of Notch targets Hes1
, and Nrarp
LSK hematopoietic progenitors, with no appreciable alteration in Hes5
, or Notch2
expression. The reason for distinct effects of Mtg16
deletion on expression of Notch target genes is unknown. One plausible explanation is that distinct repression complexes are assembled on the promoters of genes regulated by Notch, and these complexes are differentially sensitive to the loss of MTG16 or permit compensation by other MTG family proteins. Alternatively, altered Notch target gene expression in Mtg16−/−
progenitors may reflect more complex compensatory mechanisms established to preserve homeostasis in the context of Mtg16
nullizygosity. From this perspective, it is notable that MTG16 binds HDAC6 and HDAC8, in addition to HDAC1, HDAC2, and HDAC3, which MTG8 binds also. Likewise, while MTG8 binds both NCoR and SIN3A, MTG16 fails to bind the latter (3
). These findings suggest that MTG16 contributes selectively to basal repression of canonical Notch target genes and that compensation by other MTG family members might alter transcriptional outcomes. Furthermore, binding domains for SHARP and CSL map to distinct regions in MTG proteins. Our results do not exclude an indirect interaction between CSL and MTG16, mediated by an NHR3 binding protein. However, it is attractive to speculate that CSL, SHARP, and MTG family proteins form a ternary complex that, in concert with other corepressors and HDACs, limits promiscuous expression of Notch target genes. In joining the Notch transcription complex, intracellular domains of Notch receptors must overcome this repression.
Before Notch signaling is activated, CSL and Notch receptors are segregated to the nucleus and plasma membrane, respectively. Notch-activating stimuli induce proteolytic cleavage of the Notch receptor, liberating the N-ICD to permit its interaction with CSL in the nucleus. N-ICD binding to CSL overcomes transcriptional repression and initiates assembly of the activation-competent Notch transcription complex. To trigger this conversion, we hypothesized that N-ICDs would bind components of CSL repressor complexes. Utilizing a candidate approach, we characterized interactions between the N1-ICD and repressor complex proteins to discover a robust interaction with MTG16. Indeed, MTG16 interacts with the intracellular domains of all mammalian Notch receptors, suggesting a generalized relationship between it and Notch signaling. The N1-ICD binding motif in MTG16 is localized to the PST1 region, distinct from NHR domains and flanking structures responsible for interactions with DNA binding proteins, corepressors, and HDACs. No other proteins have been identified that bind this domain in MTG16.
N1-ICD expression disrupts the MTG16-CSL and MTG16-NCoR interactions. However, it fails to bind NCoR, SIN3A, or HDAC3 and fails to disrupt the interaction between CSL and NCoR (see Fig. S3 in the supplemental material). Combined, these data suggest that N1-ICD is insufficient to overcome the CSL-NCoR interaction and instead may act through MTG16 to expel NCoR from CSL repressor complexes. Notably, Kao et al. have shown that CSL interacts with SMRT and that SMRT disrupts the CSL—N1-ICD interaction and antagonizes N1-ICD-dependent transcription via CSL (26
). These data suggest that SMRT binding by CSL is favored over its interaction with N1-ICD and that N1-ICD-dependent disruption of the CSL repressor complex must be facilitated by other factors. MTG16 demonstrates characteristics consistent with this role and, in so doing, could both contribute to basal repression at CSL-regulated promoters and facilitate the transition to transcriptional activation in response to N1-ICD binding.
A hypothetical model reflecting these roles for MTG16 in the Notch transcription complex is presented in Fig. . By interacting with CSL, corepressors, and HDACs, MTG16 would reinforce repression of Notch target genes in the basal state. Notch activation would liberate the N-ICD for nuclear entry, permitting its interaction with MTG16 and causing conformational changes that destabilize repressor complex interactions. Whether N-ICD-MTG16 binding is sufficient to disrupt repressor complex integrity is not yet known. It may be that interactions between N-ICD and other repressor complex components are also required. Additionally, our data do not exclude the possibility that MTG16 remains an integral component of the Notch transcription complex despite attenuated CSL binding. In support of this notion, MTG16 interacts directly with N-ICDs and they can be found together in high molecular weight complexes in which CSL is not readily identified. To clarify these issues will require an understanding of the spatiotemporal relationships between MTG16 and Notch transcription complex components. Understanding these dynamic relationships may illuminate as-yet-undefined options for regulating responses driven by physiologic and pathological Notch activation.
FIG. 10. Model for MTG16-mediated regulation of the canonical Notch transcription complex. In the unstimulated state, MTG16, represented as an antiparallel tetramer, serves as a scaffold for corepressors (Corep's) and HDACs to repress Notch target gene expression. (more ...)
Because CSL and N-ICDs constitute core components of the canonical Notch transcription complex, our observations intimate an important relationship between MTG family proteins and Notch-dependent phenotypes. This notion is supported by ex vivo fate specification of hematopoietic progenitors in response to Notch-activating stimuli. Expression of T-cell lineage markers CD4 and CD8, normally promoted by Notch signal activation, is impaired in Mtg16−/− hematopoietic progenitors and restored by enforced expression of MTG16. However, an MTG16 mutant deficient in N1-ICD binding (MTG16Δ2C) fails to restore T-cell lineage marker expression in the context of Notch activation. This mutant protein retains each of the NHR regions in MTG16, preserving binding sites for DNA binding proteins, corepressors, and HDACs. These findings not only implicate MTG16 functionally in Notch-dependent hematopoietic lineage decisions but also underscore the importance of the N1-ICD binding region to this effect. Furthermore, the fact that MTG16 functions are important for a Notch-dependent outcome intimates a possible role in T-cell acute lymphoblastic leukemia, where mutations affecting the Notch signaling pathway are found in approximately 60% of patients.
Yet, it has been their contributions to AML pathogenesis that have motivated studies of the MTG family, and we now appreciate their central role in specifying cell fate. MTG family members interact with multiple DNA binding proteins implicated in control of hematopoiesis. Among these are E2A/HEB (16
), ZNF652 (31
), GFI1 (34
) and GFI1B (46
), PLZF (35
), TAL1/SCL (46
), TCF4 (37
), LDB1 (19
), BCL6 (9
) and GATA1 (20
). Disregulated function of one or more of these factors could contribute to the fate-specifying defect seen in Mtg16−/−
progenitors. Alternatively, Notch-dependent lineage allocation could require cooperation with these or other systems regulated by MTG16. This raises the possibility that MTG proteins may provide platforms to integrate Notch signals with other transcription factors regulated by MTG family members. Because the N-ICD and CSL binding domains are retained in proleukemic fusion proteins generated from t(8;21) and t(16;21) translocations, these interactions may be important in AML pathogenesis. These speculations provide a framework for future experiments to broaden our understanding of Notch signal integration in normal and malignant hematopoiesis.