Here we showed that the Notch1 locus is regulated by a combination of transcriptional promoters and enhancers supporting a feed-forward loop that likely augments Notch signaling at appropriate stages of T cell differentiation and leukemic transformation. Ikaros is one of the key regulators in this process working at the epigenetic level to limit recruitment of transcription-related factors.
The presence of alternative promoters at the Notch1
locus was first revealed by studies in leukemic cells from mice with combined mutations in Ikzf1
and the Notch1
canonical promoter. Although activity of the alternative Notch1
promoters was augmented in the Notch1-/-Ikzf1+/-
leukemic cells, it was also elevated in other leukemia models in which the canonical Notch1
promoter was intact. Nonetheless, combined deletion of the Notch1
canonical promoter and Ikzf1
accelerated this process, indicating participation of additional negative regulatory factors acting through the deleted region. A likely candidate is E2A, because it binds at the canonical Notch1
promoter (Yashiro-Ohtani et al., 2009
) and has similar effects as Ikaros on Notch1 transcriptional regulation and leukemogenesis. These studies also show that in T cells, the Notch1
exon1 deletion is not a null allele, particularly under conditions where active Ikaros repression is relaxed. Although it is possible that this allele is an effective null in other contexts, it will be important to assess whether deletion of negative regulatory elements and de-repression of these ligand-independent isoforms occurs in other cell types.
In addition to the canonical promoter, two alternative Notch1 promoter regions were mapped at the 5’ and at an intragenic location. Transcriptional analysis of the Notch1 locus during normal T cell development indicated that at the DN3 stage both the 5’ alternative and canonical promoters were active, whereas at the DP stage only the canonical promoter retained activity. Epigenetic studies in DP thymocytes confirmed the presence of permissive chromatin at the active promoter but also indicated that the inactive 5’ alternative promoter was in a permissive chromatin configuration possibly poised for future transcriptional induction upon activation of Notch signaling. In contrast to the 5’ promoters, the intragenic promoter region was transcriptionally repressed through the DN3 to DP transition, was not marked for transcriptional activation at the chromatin level and displayed extensive Ikaros binding.
During loss of Ikaros-mediated leukemogenesis, all three sets of Notch1 promoters acquired more permissive chromatin prior to activation of Notch signaling. Increase in permissive chromatin was detected at the 5’ alternative and canonical promoters and most notably at the intragenic promoters in pre-leukemic thymocytes on an intact Notch1 locus. The increase in chromatin accessibility at the 5’ Notch1 promoters correlated with an increase in basal transcription detected at the pre-leukemic state that preceded the strong transcriptional induction detected upon ICN accumulation at the leukemic state. Importantly, inhibition of Notch signaling, through γ-secretase inhibition or by Rbpj inactivation, interfered with the robust transcriptional induction at both the canonical and alternative promoters in leukemic cells but did not alter the increase in basal transcription observed in pre-leukemic thymocytes.
Both alternative promoters were responsible for the production of proteins that signal in limiting ligand. The full-length Notch1 protein generated by the E1a promoter lacks the signal peptide present in the canonical unprocessed form and appears to be processed differently. Notably, it is constitutively cleaved by ADAM metalloproteinases (S2) and γ-secretase (S3) in limiting ligand conditions. Thus, alternative 5’ promoter and exon usage may dictate an alternative route of cell trafficking and receptor processing that enhances Notch signaling that is pending further investigation. A range of short Notch1 protein isoforms was produced by the intragenic promoter region. A major isoform was translated from a site downstream of the S2 or ADAM site that lacked the ligand binding and NRR domains, and was a potent activator of the Notch transcriptional response.
Given these data we propose the presence of a feed-back loop in Notch signaling supported by a network of epigenetic and transcriptional regulators and Notch receptors with differential ligand-dependence for activity. Local chromatin at the Notch1
locus controls access to the basal transcription machinery and to enhancer proteins that regulate this process. The presence of Ikaros at binding sites located in proximity to all three Notch1
promoters is responsible for restricting chromatin. Ikaros association with negative chromatin remodeling factors such as Mi-2β and HDACs may restrict access or activity of positive chromatin regulators such as MLL and HATs also recruited to these sites (Kim et al., 1999
; Sridharan and Smale, 2007
). Loss of Ikaros relieves chromatin restriction increasing access to the basal transcription machinery at both ligand-dependent and -independent Notch1
promoters. This precipitates a forbidden increase in ICN, which even if present at low amounts is more effectively recruited to its target sites that are more accessible due to Ikaros removal. ICN is a potent transcriptional enhancer that can function from at least two regions in the Notch1
locus. One is at the promoter-distal IkBSN1, and the second is at an extended region downstream of the IkBSN5 in E25. Importantly, this ICN binding region shows extensive overlap with the de novo
intragenic islands of permissive chromatin arising in Ikaros mutant pre-leukemic cells. Interactions of ICN with HATs (Wallberg et al., 2002
) at its target sites may further impact transcription initiation. Transcription of the Notch1
locus is likely effected by a number of regulatory factors. In addition to Ikaros and E2A, our preliminary studies indicate a similar aberrant activation of alternative Notch1
promoters in abnormally expanding DP in an activated Akt2 transgenic model (data not shown) (Malstrom et al., 2001
). Thus, both nuclear and signaling factors implicated in leukemogenesis may participate in the regulation of this feed-forward loop in Notch signaling.
Stage-specific activation of the Notch1
promoters may be one key for modulating levels of Notch signaling during development and leukemogenesis. The induction of promoters expressing isoforms with differential ligand requirement may support a feed-forward mechanism that augments Notch signaling required for expansion of immature thymocytes. At the DN3 stage, this may be jump-started by the ligand-dependent canonical Notch1 E1c isoform and propagated by the more active E1a isoform. Subsequently at the DP stage, the E1a promoter is repressed and only the canonical promoter remains modestly active. Together with limited ligand availability, this may restrict Notch signaling to levels supporting cell survival and not proliferation at this stage of differentiation (Laky and Fowlkes, 2008
). In the Notch1
–deficient cells, deletion of the canonical promoter and lack of expression of the ligand-dependent Notch1 isoform prevented activation of the 5’ alternative promoter under conditions of restrictive chromatin. However, upon loss of Ikaros (and possibly E2A), aberrant increase in chromatin accessibility augments basal transcription at the alternative Notch1
promoters, marking transition to a pre-leukemic state. This allows a progressive increase in ICN accumulation that eventually causes robust induction of both ligand-dependent and -independent Notch1
promoters, thus sealing the transition to the leukemic state. Aberrant activation of the Notch1
promoters collaborates with Notch1 mutations that target the PEST domain in the mouse genetic systems studied here to exacerbate the leukemia phenotype. It will be important to determine whether such a mechanism is also manifested in human leukemia where a predominance of mutations in the heterodimerization and PEST domains has been described. Strong cross-species conservation of both the canonical and alternative promoters gives support. Targeting the transcriptional regulation of Notch1
may open new avenues for leukemia treatment. Small molecules targeting enzymes that control chromatin accessibility in combination with γ-secretase inhibitors may provide better treatment protocols for curing T-ALL.