CCAAT enhancer-binding protein beta regulates constitutive gene expression during late stages of monocyte to macrophage differentiation.

Human monocyte to macrophage differentiation is accompanied by pronounced phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are poorly understood. Here, we studied the regulation of the macrophage-specific chitotriosidase (CHIT1) gene promoter to gain insights into the mechanisms of transcriptional control during the differentiation of human blood monocytes into macrophages. We used transient transfections to define a cell type-specific minimal promoter that was mainly dependent on a proximal C/EBP motif that bound multiple C/EBP factors in gel shift assays. In depth analysis of occupied promoter elements using in vivo footprinting and chromatin immunoprecipitation analyses demonstrated the differentiation-associated recruitment of C/EBPbeta and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBPbeta promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBPbeta protein during the differentiation process, whereas C/EBPbeta mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBPbeta in differentiated macrophages but not in human blood monocytes.

Transcriptional regulation of CHI3L1, a marker gene for late stages of macrophage differentiation.

The protein product of the CHI3L1 gene, human cartilage 39-kDa glycoprotein (HC-gp39), is a tissue-restricted, chitin-binding lectin and member of glycosyl hydrolase family 18. In contrast to many other monocyte/macrophage markers, its expression is absent in monocytes and strongly induced during late stages of human macrophage differentiation. To gain insights into the molecular mechanisms underlying its cell type-restricted and maturation-associated expression in macrophages, we initiated a detailed study of the proximal HC-gp39 promoter. Deletion analysis of reporter constructs in macrophage-like THP-1 cells localized a region directing high levels of macrophage-specific reporter gene expression to approximately 300 bp adjacent to the major transcriptional start site. The promoter sequence contained consensus binding sites for several known factors, and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1, and C/EBP proteins was detectable in gel shift assays. In vivo footprinting assays with dimethyl sulfate demonstrate that the protection of corresponding sequences was enhanced in macrophages compared with monocytes. Mutational analysis of transcription factor binding sites indicated a predominant role for a single Sp1 binding site in regulating HC-gp39 promoter activity. In addition, gel shift assays using nuclear extracts of monocytes and macrophages demonstrated that the binding of nuclear Sp1, but not Sp3, markedly increases during macrophage differentiation. Our results further highlight the important role of Sp1 in macrophage gene regulation.