AUTHOR=Gu Xiaoyu , Qiao Xue , Yu Simiao , Song Xiaorui , Wang Lingling , Song Linsheng 

TITLE=Histone lysine-specific demethylase 1 regulates the proliferation of hemocytes in the oyster Crassostrea gigas

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.1088149

DOI=10.3389/fimmu.2022.1088149

ISSN=1664-3224

ABSTRACT=<sec><title>Background</title><p>Lysine-specific demethylase 1 (LSD1) is an essential epigenetic regulator of hematopoietic differentiation, which can specifically mono-methylate H3K4 (H3K4me1) and di-methylate H3K4 (H3K4me2) as a transcriptional corepressor. Previous reports have been suggested that it participated in hematopoiesis and embryonic development process. Here, a conserved LSD1 (<italic>Cg</italic>LSD1) with a SWIRM domain and an amino oxidase (AO) domain was identified from the Pacific oyster <italic>Crassostrea gigas</italic>.</p></sec><sec><title>Methods</title><p>We conducted a comprehensive analysis by various means to verify the function of <italic>Cg</italic>LSD1 in hematopoietic process, including quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, immunofluorescence assay, RNA interference (RNAi) and flow cytometry.</p></sec><sec><title>Results</title><p>The qRT-PCR analysis revealed that the transcripts of <italic>Cg</italic>LSD1 were widely expressed in oyster tissues with the highest level in the mantle. And the transcripts of <italic>Cg</italic>LSD1 were ubiquitously expressed during larval development with the highest expression level at the early D-veliger larvae stage. In hemocytes after <italic>Vibrio splendidus</italic> stimulation, the transcripts of <italic>Cg</italic>LSD1 were significantly downregulated at 3, 6, 24, and 48 h with the lowest level at 3 h compared to that in the Seawater group (SW group). Immunocytochemical analysis showed that <italic>Cg</italic>LSD1 was mainly distributed in the nucleus of hemocytes. After the <italic>Cg</italic>LSD1 was knocked down by RNAi, the H3K4me1 and H3K4me2 methylation level significantly increased in hemocyte protein. Besides, the percentage of hemocytes with EdU-positive signals in the total circulating hemocytes significantly increased after <italic>V. splendidus</italic> stimulation. After RNAi of <italic>Cg</italic>LSD1, the expression of potential granulocyte markers <italic>Cg</italic>SOX11 and <italic>Cg</italic>AATase as well as oyster cytokine-like factor <italic>Cg</italic>Astakine were increased significantly in mRNA level, while the transcripts of potential agranulocyte marker <italic>Cg</italic>CD9 was decreased significantly after <italic>V. splendidus </italic> stimulation.</p></sec><sec><title>Conclusion</title><p>The above results demonstrated that <italic>Cg</italic>LSD1 was a conserved member of lysine demethylate enzymes that regulate hemocyte proliferation during the hematopoietic process.</p></sec>