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

Lisa Frankenreiter,Hannes Schmid,Anja C Nagel,Philipp Hoffmeister,Mirjam Zimmermann,Bernd M Gahr,Tilman Borggrefe,Franz Oswald,Aleksandra Turkiewicz,Sebastian Deichsel

doi:10.3389/fcell.2021.658820

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.

Highlights

Simple signaling transduction mechanisms can induce a surprisingly large variety of cell types and organs and form the basis to understand regulatory networks governing development in higher organisms
We showed that Su(H) is phosphorylated at Serine 269 in Drosophila S2 cells, and that the introduction of a negative charge at this position interfered with the DNAbinding capability and transcriptional activity of Su(H)
As Notch signaling activity participates in a multitude of cell fate decisions, perturbation of the signal transmission as well as signal strength is linked to a multitude of human diseases, FIGURE 5 | N-dependent lymph gland development is responsive to Su(H) phospho-site mutations. (A–F) Shown are primary lobes of third instar larval lymph glands stained with anti-Hnt and anti-Pzg

Summary

Introduction

Simple signaling transduction mechanisms can induce a surprisingly large variety of cell types and organs and form the basis to understand regulatory networks governing development in higher organisms. Notch is required for the generation of hematopoietic stem cells and controls lymphoid cell fates, i.e., T-cell commitment (Bigas and Espinosa, 2012). The latter conceptually parallels the role of Notch in Drosophila hematopoiesis, where it plays an instructive role in the differentiation of crystal cells (Duvic et al, 2002). Notch signaling acts upstream of lz as well as in conjunction with Lz to promote crystal cell precursor maintenance, specification and differentiation (Lebestky et al, 2003; Terriente-Felix et al, 2013; BlancoObregon et al, 2020). Notch activity is required continuously within the crystal cell to promote maturation, cell growth and survival (Mukherjee et al, 2011; Terriente-Felix et al, 2013; Miller et al, 2017)

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