Page 39 - Haematologica August 2018

CARD10 regulates granulocytic differentiation
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Figure 5. Knock down of CARD10 impairs myeloid differentiation. (A) RT-PCR analysis of CARD10 expres- sion in human NB4 cells transduced with either non-target shRNA (NT) or CARD10 specific shRNAs (sh4 and sh5). Y-axis represents relative expression of CARD10 normalized to transcript levels of GAPDH (B) Proportion of CD11b+ NB4 cells sta- bly transduced with either control (NT) or CARD10 shRNA and cultured with 1 μM ATRA for different dura- tion. (C) RT-PCR validation of Card10 knock-down in murine Lin–Kit+ bone marrow (BM ) cells. Y-axis represents relative expression of Card10 nor- malized to Gapdh. (D) Granulocytic differentiation of Lin–Kit+ BM cells stably expressing either NT or Card10 shRNAs (sh2 or sh5) in response to 10ng/mL of GM-CSF. Differentiation was monitored by flow cytometric quantification of CD11b+Gr1+ cells. Results are aver- age of three independent experi- ments. *P<0.05, **P<0.01.
Supplementary Table S2). Furthermore, GSEA analysis of downregulated genes revealed a strong enrichment for genes involved in myeloid development (Figure 6C). Expression signature of Card10 knock-down Lin–Kit+ BM cells (carried out with two independent shRNAs) was compared to that of Cebpe KO immature granulocytes. Twenty-nine of the 94 genes downregulated in Card10 knock-down cells were also downregulated in Cebpe KO cells (Figure 6D). We validated the lower expression of prominent myeloid specific genes using RT-PCR in Card10 KD Lin–Kit+ BM cells (Figure 6E). These results show that loss of CARD10 affects expression of genes implicated in myelopoesis.
Discussion
CEBPE is a transcription factor essential for functional maturation of granulocytes. Patients with neutrophil-spe- cific granule deficiency have mutations of the CEBPE gene.15,16 Cebpe knockout mice recapitulate the disease and fail to produce terminally differentiated granulocytes. Previous studies from our group and others have identi- fied several key targets of CEBPE,23-26 including a compre- hensive approach that curated a list of CEBPE targets by comparing CEBPE binding sites to the gene expression changes in sorted granulocytes from Cebpe WT and KO cells.21 The present study extends the analysis to identify additional novel targets of CEBPE including genes with intronic or intergenic binding. Analysis of histone modifi- cations at loci of genes with intronic/intergenic peaks showed that a majority of them had an active epigenetic signatures in the granulocytic population, suggesting these
Knock-down of CARD10 impairs granulocytic differentiation of murine Lin–Kit+ BM cells
We evaluated further the role of CARD10 in granulocyt- ic differentiation. shRNAs targeting murine Card10 were initially screened in NIH/3T3 cells (Online Supplementary Figure S6A) and two shRNAs that silenced Card10 were used to transduce murine Lin–Kit+ bone marrow (BM) cells. These shRNA sequences also robustly reduced the expres- sion of Card10 in murine Lin–Kit+ BM cells (Figure 5C). Lin–Kit+ myeloid progenitors were transduced with either control or mouse Card10 shRNAs and grown in the pres- ence of IL3, IL6, SCF and GM-CSF. The proportion of CD11b+Gr1+ cells were analysed at different time points using flow cytometry. Our analysis revealed that Card10 knock-down resulted in significantly lower proportion of granulocytes at all the time points tested (control shRNA: 50%, Card10 KD: 19% (sh2) and 11% (sh5) at 7 days) (Figure 5D and Online Supplementary Figure S6B). This fur- ther illustrated a role for CARD10 in myeloid cell differen- tiation.
Knock-down of Card10 affects expression of myeloid-specific genes
To understand the changes in gene expression following Card10 knock-down, RNA-Seq of bulk control and Card10 knock-down Lin–Kit+ BM cells was performed. Ninety- four genes were downregulated and 68 genes were upreg- ulated following Card10 knock-down (FDR<0.1) (Figure 6A). Gene Ontology analysis of differentially expressed genes revealed enrichment of genes involved in immune response, inflammatory response, leukocyte migration and response to external stimuli (Figure 6B and Online
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