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

ABSTRACT: 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.

SUBMITTER: Frankenreiter L

PROVIDER: S-EPMC8079769 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe Notch signaling pathway is fundamental to the regulation of many cell fate decisions in eumetazoans. Not surprisingly, members of this pathway are highly conserved even between vertebrates and invertebrates. There is one notable exception, Hairless, which acts as a general Notch antagonist in Drosophila. Hairless silences Notch target genes by assembling a repressor complex together with Suppressor of Hairless [Su(H)] and the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). Now with the availability of genomic databases, presumptive Hairless homologues are predicted, however only in insect species. To further our understanding of Hairless structure and function, we have cloned the Hairless gene from Apis mellifera (A.m.H) and characterized its functional conservation in Drosophila.ResultsThe Apis Hairless protein is only one third of the size of the Drosophila orthologue. Interestingly, the defined Suppressor of Hairless binding domain is interrupted by a nonconserved spacer sequence and the N-terminal motif is sufficient for binding. In contrast to Apis Hairless, the Drosophila orthologue contains a large acidic domain and we provide experimental evidence that this acidic domain is necessary to silence Hairless activity in vivo. Despite the dramatic size differences, Apis Hairless binds to the Drosophila Hairless interactors Su(H), Gro, CtBP and Pros26.4. Hence, Apis Hairless assembles a repressor complex with Drosophila components that may have a different topology. Nevertheless, Apis Hairless is sufficient to repress the Notch target gene vestigial in Drosophila. Moreover, it is able to rescue Hairless mutant phenotypes, providing in vivo evidence for its function as a bona fide Notch antagonist.ConclusionThis is the first interspecies-complementation analysis of the Hairless gene. Guided by evolutionary comparisons, we hope to eventually identify all the relevant structural domains and cofactors of Hairless, thereby opening an avenue for further insights into the repressor-complexes that down-regulate Notch signaling also in other, higher eukaryotes.

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

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