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 RBPJS221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJS221A 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.

Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila

“…core Notch response, N-only, NS-only). Indeed, recent reports point towards different post-translational modifications for Su(H) (Frankenreiter et al, 2021). However, whether they lead to different transcriptional programs, and whether they occur in vivo in the N and NS models, remain to be studied.…”

A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects

“…Intriguingly, an analogous cellular mechanism that compromises Su(H) DNA binding has previously been identified in Drosophila 33,34 . In this case, phosphorylation of Su(H) residue S269, which interacts with DNA, blocks DNA binding, and plays a role in fly hematopoiesis.…”

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

“…Having shown the RBPJL-SHARP interaction, we wanted to characterize the functional consequences and determine the repressive capacity of RBPJL at endogenous Notch target genes. As we previously found that the deletion of RBPJ results in the derepression of Notch target genes in a mature T cell line [ 19 ] and in HeLa cells [ 48 ], we now performed rescue experiments re-expressing RBPJ or RBPJL fused to GFP using RBPJ-depleted HeLa cells ( Figure 7 A,B).…”