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Analysis of Potential Non-Canonical or Alternate STAT5 Functions in Immune Development and Growth
Nagendra Awasthi1, Alister C Ward1,2, Clifford Liongue1,2
1School of Medicine, Deakin University, 3216 Victoria, Australia.
Background:
Signal transducer and activator of transcription (STAT) proteins play key roles in development, growth, and homeostasis. These roles have principally been assigned to their "canonical" function as inducible transcriptional activators acting downstream of cytokines and other factors. However, variant "non-canonical" functions have also been identified. The potential in vivo role for non-canonical STAT functions was investigated in the zebrafish model.
Methods:
Two zebrafish Stat5.1 mutants were generated using CRISPR/Cas9 that should impact canonical functionality: one with a deleted transactivation domain (ΔTAD) and another with a disrupted tyrosine motif (ΔTM). Immune cell development, growth, and adiposity of these Stat5.1 mutants were assessed in comparison to a Stat5.1 knockout (KO) mutant in which both canonical and non-canonical functions were ablated.
Results:
Both the ΔTAD and ΔTM mutants showed significantly reduced embryonic T lymphopoiesis, similar to the KO mutant. Additionally, adult ΔTAD and ΔTM mutants displayed a decrease in T cell markers in the kidney, but not as severe as the KO, which also showed T cell disruption in the spleen. Severe growth deficiency and increased adiposity were observed in all mutants, but ΔTAD showed a more modest growth defect whereas ΔTM exhibited more profound impacts on both growth and adiposity, suggesting additional gain-of-function activity.
Conclusions:
These results indicate that canonical Stat5.1 plays a major role in T cell development and growth throughout the lifespan and non-canonical Stat5.1 functions also contribute to aspects of adult T lymphocyte development and growth, with alternate functions impacting growth and adiposity.
Insights
Canonical and non-canonical Signal transducer and activator of transcription (STAT) 5.1 functions are vital for zebrafish T cell development and growth. Non-canonical STAT5.1 functions also influence adult T lymphocyte development and adiposity.
Area of Science:
- Molecular Biology
- Developmental Biology
- Immunology
Background:
- Signal transducer and activator of transcription (STAT) proteins are crucial for development, growth, and homeostasis.
- Their primary role is canonical transcriptional activation downstream of cytokines.
- Emerging evidence suggests variant non-canonical functions for STAT proteins.
Purpose of the Study:
- To investigate the in vivo role of non-canonical STAT5.1 functions in zebrafish.
- To differentiate the roles of canonical versus non-canonical STAT5.1 activity.
Main Methods:
- Generated zebrafish Stat5.1 mutants using CRISPR/Cas9 targeting canonical domains (transactivation domain - ΔTAD, tyrosine motif - ΔTM).
- Assessed immune cell development, growth, and adiposity in comparison to a Stat5.1 knockout (KO) mutant.
- Analyzed T lymphopoiesis, T cell markers in kidney and spleen, growth, and adiposity.
Main Results:
- Both ΔTAD and ΔTM mutants exhibited reduced embryonic T lymphopoiesis, similar to the KO mutant.
- Adult mutants showed decreased T cell markers in the kidney; the KO mutant also had spleen T cell disruption.
- All mutants displayed growth deficiency and increased adiposity, with ΔTM showing more severe impacts, suggesting gain-of-function activity.
Conclusions:
- Canonical Stat5.1 is essential for T cell development and growth throughout life.
- Non-canonical Stat5.1 functions contribute to adult T lymphocyte development and growth.
- Distinct non-canonical STAT5.1 functions impact growth and adiposity.
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