Transcription Factor RBPJL Is Able to Repress Notch Target Gene Expression but Is Non-Responsive to Notch Activation.

The Notch signaling pathway is an evolutionary conserved signal transduction cascade present in almost all tissues and is required for embryonic and postnatal development, as well as for stem cell maintenance, but it is also implicated in tumorigenesis including pancreatic cancer and leukemia. The transcription factor RBPJ forms a coactivator complex in the presence of a Notch signal, whereas it represses Notch target genes in the absence of a Notch stimulus. In the pancreas, a specific paralog of RBPJ, called RBPJL, is expressed and found as part of the heterotrimeric PTF1-complex. However, the function of RBPJL in Notch signaling remains elusive. Using molecular modeling, biochemical and functional assays, as well as single-molecule time-lapse imaging, we show that RBPJL and RBPJ, despite limited sequence homology, possess a high degree of structural similarity. RBPJL is specifically expressed in the exocrine pancreas, whereas it is mostly undetectable in pancreatic tumour cell lines. Importantly, RBPJL is not able to interact with Notch-1 to -4 and it does not support Notch-mediated transactivation. However, RBPJL can bind to canonical RBPJ DNA elements and shows migration dynamics comparable to that of RBPJ in the nuclei of living cells. Importantly, RBPJL is able to interact with SHARP/SPEN, the central corepressor of the Notch pathway. In line with this, RBPJL is able to fully reconstitute transcriptional repression at Notch target genes in cells lacking RBPJ. Together, RBPJL can act as an antagonist of RBPJ, which renders cells unresponsive to the activation of Notch.

Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila.

The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser269 impedes DNA-binding. By genome-engineering, we now introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H) S269A ] or a phospho-mimetic [Su(H) S269D ] isoform. Su(H) S269D mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H) S269A mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJ S221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJ S221A rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.

Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription.

Activation and repression of Notch target genes is mediated by transcription factor CSL, known as Suppressor of Hairless (Su(H)) in Drosophila and CBF1 or RBPJ in human. CSL associates either with co-activator Notch or with co-repressors such as Drosophila Hairless. The nuclear translocation of transcription factor CSL relies on co-factor association, both in mammals and in Drosophila. The Drosophila CSL orthologue Su(H) requires Hairless for repressor complex formation. Based on its role in transcriptional silencing, H protein would be expected to be strictly nuclear. However, H protein is also cytosolic, which may relate to its role in the stabilization and nuclear translocation of Su(H) protein. Here, we investigate the function of the predicted nuclear localization signals (NLS 1-3) and single nuclear export signal (NES) of co-repressor Hairless using GFP-fusion proteins, reporter assays and in vivo analyses using Hairless wild type and shuttling-defective Hairless mutants. We identify NLS3 and NES to be critical for Hairless function. In fact, H⁎NLS3 mutant flies match H null mutants, whereas H⁎NLS3⁎NES double mutants display weaker phenotypes in agreement with a crucial role for NES in H export. As expected for a transcriptional repressor, Notch target genes are deregulated in H⁎NLS3 mutant cells, demonstrating nuclear requirement for its activity. Importantly, we reveal that Su(H) protein strictly follows Hairless protein localization. Together, we propose that shuttling between the nucleo-cytoplasmic compartments provides the possibility to fine tune the regulation of Notch target gene expression by balancing of Su(H) protein availability for Notch activation.