Abstract
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.
Highlights
(C/EBP)2, and core binding factor (CBF) families and several other transcription factors [2, 3]
The current study demonstrates that the transcription factor C/EBP, expressed at comparable levels during the monocytic differentiation process, is increasingly phosphorylated at Thr-235, suggesting that human macrophages contain high constitutive levels of functional C/EBP
Several studies using cell lines or C/EBP-deficient macrophages established that C/EBP contributes to the inducible expression of several inflammatory genes including interleukin (IL) 1, IL-12, IL-6 or PTGS2 in mouse macrophages [46, 47]
Summary
C/EBP, CCAAT enhancer-binding protein; CSF-1, colony-stimulating factor 1; DMS, dimethyl sulfate; PMA, phorbol 12-myristate 13-acetate; RACE, rapid amplification of cDNA ends; LAP, liver-activating protein; LIP, liver-inhibitory protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. The specific biological role of both chitinase family members, is difficult to study since both genes appear to be differentially regulated in primates and rodents [30, 31]. The similar expression profile in human monocyte-derived macrophages in conjunction with their close proximity on chromosome 1 suggests common regulatory mechanisms for both chitinase family genes. The current study investigates the structure and regulation of the proximal CHIT1 promoter as well as the binding and recruitment of transcription factors at the proximal promoter region of CHIT1 during monocyte to macrophage differentiation. We show that the differentiationassociated CHIT1 induction is accompanied by the binding of transcription factor PU. and, most strikingly, of C/EBP to the proximal CHIT1 gene promoter. Whereas C/EBP total mRNA or protein levels remain largely unchanged, we show that C/EBP recruitment to its target gene promoter is preceded by an increase in phosphorylation of nuclear C/EBP during the differentiation process
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