The interaction of Pu.1 and cMyb in zebrafish neutrophil development

Granulopoiesis is a highly ordered and precisely regulated process in which hematopoietic-related transcription factors play crucial roles. These transcription factors form complex regulatory networks through interactions with their co-factors or with each other, and anomalies in these networks can lead to the onset of leukemia. While the structures and functions of dozens of transcription factors involved in this process have been extensively studied, research on the regulatory relationships between these factors remains relatively limited. PU.1 and cMYB participate in multiple stages of neutrophil development, and their abnormalities are often associated with hematologic disorders. However, the regulatory relationship between these factors in vivo and their mode of interaction remain unclear. In this study, zebrafish models with cMyb overexpression (cmyb^hyper) and Pu.1 deficiency (pu.1^G242D/G242D) were utilized to systematically investigate the interaction between Pu.1 and cMyb during granulopoiesis through whole-mount in situ hybridization, qRT-PCR, fluorescence reporting systems, and rescue experiments. The results showed a significant increase in cmyb expression in neutrophils of the pu.1^G242D/G242D mutant, while there was no apparent change in pu.1 expression in cmyb^hyper. Further experiments involving injection of morpholino (MO) to decrease cmyb expression in pu.1^G242D/G242D mutants, followed by SB and BrdU staining to assess neutrophil quantity and proliferation, revealed that reducing cmyb expression could rescue the abnormal proliferation phenotype of neutrophils in the pu.1^G242D/G242D mutant. These findings suggest that Pu.1 negatively regulates the expression of cMyb during neutrophil development. Finally, through the construction of multi-site mutation plasmids and a fluorescent reporter system, confirmed that Pu.1 directly binds to the +72 bp site in the cmyb promoter, exerting negative regulation on its expression. In conclusion, this study delineates that Pu.1 participates in neutrophil development by regulating cmyb expression. This provides new insights into the regulatory relationship between these two factors and their roles in diseases.