Representational difference analysis using myeloid cells from C/EBPε deletional mice

Abstract

C/EBPε is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue specific manner; it is expressed exclusively in myeloid cells. C/EBPε-deficient mice developed normally but failed to generate functional neutrophils and eosinophils, and these mice died of opportunistic infections suggesting that C/EBPε may play a central role in myeloid differentiation. To identify myelomonocytic genes regulated by the C/EBPε gene, we performed representational difference analysis (RDA), a polymerase chain reaction (PCR)-based subtractive hybridization using neutrophils and macrophages from wild-type and C/EBPε knockout mice. We identified a set of differentially expressed genes, including chemokines specific to myelomonocytic cells. Several novel genes were identified that were differentially expressed in normal myelomonocytic cells. Taken together, we have found several genes whose expression might be enhanced by C/EBPε.

Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency

“…Morphologically, the preparations for array hybridization were composed of ϳ30% monocytes, 60% neutrophils, and 7-10% lymphocytes in the C/EBPε ϩ/ϩ and C/EBPε Ϫ/Ϫ mice, as determined by differential counts of cytospins stained with Wright-Giemsa. This cellular composition was consistent with prior studies and would allow us to identify monocytic and granulocytic gene expression differences [10,19]. To minimize variation among individual mice, the samples were pooled according to the mouse genotype, and the chip set was hybridized.…”

“…Representational difference analysis using peritoneal neutrophils and macrophages from wild-type and C/EBPε-deficient mice identified a small set of differentially regulated genes. These included several genes specific to myelomonocytic cells [19]. Consistent with aberrant macrophage gene expression, phenotypic changes were observed in vivo.…”

C/EBPα and C/EBPɛ induce the monocytic differentiation of myelomonocytic cells with the MLL-chimeric fusion gene

“…C/EBPe also induces the gene expression of monocyte-colony-stimulating factor receptor (Williams et al, 1998), and is required for the development and function of mature macrophages (Tavor et al, 2002). The expression of chemokines MIP-1g and MCP-3 is defective in macrophages from C/EBPe-deficient mice (Kubota et al, 2000). This study indicates that HF-6 cells are presumably arrested at the monoblastic stage based on both morphological and immunophenotypical analysis.…”

The Role of C/EBPε in the Terminal Stages of Granulocyte Differentiation

“…Induction of the M‐CSF receptor with C/EBPε expression in these cells also suggests a role for C/EBPε in macrophage development and function [47]. Likewise, representational difference analysis of thioglycollate‐induced neutrophils and macrophages from C/EBPε‐deficient mice detects predominantly macrophage‐expressed genes including cathepsin L, MIP‐1γ, MCP‐3, and galactose/N‐acetylgalactosamine‐specific lectin [49]. C/EBPε induction of monocyte‐specific genes may represent a divergence between the role of C/EBPε in humans and rodents—monocytic differentiation of human bipotent cell lines such as HL60, U937, and NB4 results in reduced C/EBPε mRNA expression, and granulocytic differentiation produces increased expression [42, 43].…”