Notch receptors participate in a highly conserved signaling pathway that regulates cellular differentiation and homeostasis in a dose- and context-dependent fashion (for review, see 
). Mammals express four different Notch receptors (Notch1-4), large type I transmembrane glycoproteins composed of a series of characteristic structural motifs. Activation of Notch receptors normally depends on two successive types of proteolytic cleavages (for review, see 
). First, binding of ligands to the extracellular domain of Notch triggers proteolytic cleavage just external to the transmembrane domain by ADAM-type metalloproteases. This creates a truncated membrane-tethered form of Notch that is recognized by the gamma-secretase protease complex. Additional cleavages by gamma-secretase free the intracellular domain of Notch (ICN) from the membrane, allowing it to translocate to the nucleus and form a transcriptional activation complex with the DNA-binding factor CSL and a co-activator protein of the Mastermind-like (MAML) family.
Structural studies have provided a model for the stepwise assembly of the CSL/ICN/MAML transcriptional activation complex 
. The intracellular portions of Notch1-4 (ICN1-4) contain N-terminal RAM domains, which bind CSL with relatively high affinities 
, and 7 iterated ankyrin repeats. Under physiologic conditions, the RAM domain likely mediates the initial association of ICN and CSL, which enables formation of a CSL
ANK composite surface that recruits MAMLs, an essential event for transcriptional activation of target genes and subsequent downstream functions 
In line with their critical role in assembly of this complex, ANK domains are the most highly conserved part of ICN1-4, followed by the RAM domains (summarized in ). In contrast, sequences C-terminal of ANK are substantially more varied. At the far C-termini of ICN1-4 are PEST degron domains that stimulate ICN degradation. Between the ANK and the PEST domains lies the most divergent portion of mammalian ICN1-4. In ICN1 this region includes a strong transcriptional activation domain (TAD), whereas the analogous region of Notch2 appears to contain a weaker TAD 
. The same portion of Notch3 lacks a conventional TAD, but instead is proposed to interact with an as-of-yet unknown zinc-finger transcription factor that contributes to the activation of specific target genes, such as Hes5 
. Sequences immediately C-terminal of ANK in ICN1 and ICN2 contain phosphorylation sites that may modulate function in response to cytokines 
. The function of the region between ANK and PEST in ICN4 is unknown.
Although ICN1-4 differ in their transactivation of Notch-responsive reporter genes in transient expression assays 
, most direct comparisons performed to date in vivo
have not revealed functional differences. Replacement of the last 426 amino acids of Notch2 with the same region of Notch1 (corresponding to the seventh ankyrin repeat, the TAD, and the PEST domain) by gene targeting results in normal mice 
, suggesting that these portions of ICN1 and ICN2 are equivalent functionally. Of relevance to this report, transduced ICN1 or ICN4 both induce human hematopoietic progenitors to undergo T cell development following transplantation into NOD/SCID mice 
An important pathophysiologic outcome of ICN overexpression is neoplasia. Retroviral expression of ICN1 in hematolymphoid progenitors is a potent inducer of murine T-ALL 
, and the majority of human and murine T-ALLs harbor gain-of-function mutations in Notch1 (for recent review, see ref. 
. Feline leukemia viruses that transduce the coding sequences for the RAM and ANK domains of ICN2 accelerate T-ALL development 
, and transgenic LCK-ICN3 mice develop T-ALL with high penetrance and short latency periods 
, indicating that Notch2 and Notch3 also have leukemic potential. Recent deep sequencing studies have identified acquired mutations that result in deletion of the C-terminal PEST domain in 10-15% of human chronic lymphocytic leukemia (CLL) 
, a type of Notch1 mutation originally identified in human T-cell acute lymphoblastic leukemia (T-ALL) 
that stabilizes ICN1 and enhances the transactivation of target genes in leukemia cells. Conversely, Notch signaling has tumor suppressive effects in the context of squamous epithelium 
, a finding that emphasizes the context-dependent outcome of Notch signaling.
was first identified as a proviral insertion site in murine mammary tumors, and enforced expression of ICN4 contributes to development of adenocarcinoma 
. However, the transforming abilities of ICN1-4 have not been compared directly in vivo
in a single cellular context, and other data suggest that ICNs have divergent activities. For example, ICN1 and ICN2 reportedly have opposing effects on the growth of brain tumors 
. Thus, the physiologic and pathophysiologic interchangeability of ICN1-4 is an open question.
To address this issue, we compared the ability of ICN1-4 to drive T cell development and cause T-ALL in vivo
and to rescue T cell progenitors from blockade of endogenous Notch signaling in thymic organ culture assays. We find that while ICN1-4 all support T cell development, only ICN1-3 induce T-ALL efficiently. T cell progenitors expressing ICN4 appear to be actively extinguished and disappear by 6 months post-transplantation, a phenotype resembling that caused by “hypoleukemic” weak gain-of-function forms of Notch1 
. Further, studies performed with chimeric receptors allowed us to map the structural basis for this difference in leukemogenicity to repeats 2–7 of the ANK domain, which influence the ability of ICN to activate expression of Myc
, a key Notch target gene implicated in leukemogenesis. These studies demonstrate that the transforming activities of Notch receptors in hematolymphoid progenitors are not equivalent, and that this functional divergence is attributable in part to variation in the highly conserved ANK domains.