E3 Ubiquitin Ligase CHIP Inhibits Haemocyte Proliferation and Differentiation via the Ubiquitination of Runx in the Pacific Oyster

Mollusca first evolve primitive immune cells (namely, haemocytes), which assemble a notable complex innate immune system, which are continuously produced through proliferation and differentiation and infused in the haemolymph. As a typical E3 ligase, CHIP is critical for immune cell turnover and homeostasis in vertebrates. In this study, a CHIP homolog (<i>Cg</i>CHIP) with a high expression in haemocytes was identified in oysters to investigate its role in the proliferation and differentiation of ancient innate immune cells. <i>Cg</i>CHIP exhibited a widespread distribution across all haemocyte subpopulations, and the knockdown of <i>Cg</i>CHIP altered the composition of haemocytes as examined by flow cytometry. Mechanistically screened with bioinformatics and immunoprecipitation, a key haematopoietic transcription factor <i>Cg</i>Runx was identified as a substrate of <i>Cg</i>CHIP. Moreover, amino acids in the interacted intervals of <i>Cg</i>CHIP and <i>Cg</i>Runx were determined by molecular docking. Experimental evidence from an in vitro culture model of an agranulocyte subpopulation and an in vivo oyster model revealed that the knockdown of <i>Cg</i>CHIP and <i>Cg</i>Runx had opposing effects on agranulocyte (precursor cells) differentiation and granulocyte (effector cells) proliferation. In summary, <i>Cg</i>CHIP negatively regulated agranulocyte differentiation and granulocyte proliferation by mediating the ubiquitination and degradation of <i>Cg</i>Runx in oysters. These results offer insight into the involvement of ubiquitylation in controlling haemocyte turnover in primitive invertebrates.