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P. Shyamsunder et al.
ABC
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Figure 6. Gene expression changes in Card10 deficient murine Lin–Kit+ bone marrow cells. (A) Volcano plot depicts genes differentially expressed in Card10 knock- down Lin–Kit+ BM cells compared to the WT cells (RNA-Seq). (B and C) Gene Ontology analysis (B) and GSEA plot (C) reveals enrichment of genes involved in myeloid development. (D) Venn diagram shows overlap of downregulated genes in WT vs. Cebpe KO (sorted immature granulocytes) compared to Card10 KD Lin–Kit+ BM cells. (E) RT-PCR measuring the transcript levels of myeloid specific genes in NT and Card10 knock-down Lin–Kit+ BM cells. Y-axis represents relative expression of genes normalized to Gapdh. *P<0.05, **P<0.01, ***P<0.001.
genes are also positively regulated by CEBPE during differ- entiation of granulocytes. Amongst these, Card10 was investigated as a putative novel target of CEBPE.
We identified binding of CEBPE at two sites upstream of the Card10 gene and hypothesized that CEBPE might reg- ulate transcription of Card10. We verified occupancy of CEBPE to the -7kb region upstream of Card10 gene using ChIP-PCR, luciferase reporter assay and EMSA. We observed impaired granulocytic diffferentiation following Card10 knock-down in both human (NB4) and murine (Lin–Kit+) BM cells. These findings suggest that CARD10 may be an important mediator of granulocytic differentia- tion.
To understand how deficiency of CARD10 causes per- turbation in myeloid differentiation, we performed expression analysis of Card10 knock-down and murine Lin–Kit+ bone marrow cells. RNA-seq data showed that knock-down of Card10 affected expression of genes involved in myeloid development and function. Among the genes downregulated in Card10 knock-down cells, 29 were also downregulated in Cebpe KO granulocytes and analysis of their gene expression pattern revealed that these genes were exclusively expressed in the granulocytic population (Online Supplementary Table S3).
Migration of neutrophils to an inflammatory site is modulated by the activation of NFκB signalling.27,28 Published literature has documented a role for CARD10 as a scaffold protein for the NFκB signalling pathway, with no reference of its expression or role in granulopoiesis.19 The present study highlights that Card10 expression is
regulated by CEBPE and that Card10 deficient cells have impaired granulopoiesis accompanied by reduced tran- script levels of genes important in myeloid cell function. The impaired migration and phagocytosis observed in Cebpe KO mice may be contributed by lowered levels of Card10.
We have previously shown that stimulating activity of CEBPE in vivo can enhance the antimicrobial function of the body.29 As microbes evolve to discover new ways to become resistant to antibiotics, enhancing innate immuni- ty becomes more important. Whether the transcriptional changes observed upon Card10 knock-down is an effect of loss of mature granulocytes or a direct effect of Card10 function, warrants further analysis. In summary, this study demonstrates that Card10 is a novel CEBPE target gene and is implicated in granulocytic differentiation.
Acknowledgments
We would like to thank the staff of Comparative Medicine, NUS for their support in maintenance of mouse colonies and experiments involving mice. We would also like to acknowledge the expert help and support from the FACS facility at CSI, Singapore. We thank the Melamed Family and Reuben Yeroushalmi for their generous support.
Funding
This work was also funded by the Leukemia Lymphoma Society of America, (the Singapore Ministry of Health’s National Medical Research Council (NMRC) under its Singapore Translational Research (STaR) Investigator Award
haematologica | 2018; 103(8)