In vivo protein interaction with the mouse M-lysozyme gene downstream enhancer correlates with demethylation and gene...

Differentiation of myeloid precursor cells results in transcriptional activation of the myeloid-specific murine M-lysozyme gene. M-lysozyme gene expression depends on the differentiation state of the myeloid cells and provides a marker for myeloid leukemias. The mouse lysozyme downstream enhancer (MLDE) was colocalized previously with the DNase I hypersensitive site in the chromatin of mature macrophages and shown to be macrophage differentiation-dependent. The correlation of the hypersensitive site appearance with expression of the M-lysozyme gene suggests that the enhancer becomes activated during macrophage differentiation. However, the predominant MLDE-binding protein GABP is ubiquitously expressed, indicating that additional regulatory mechanisms are required for restricting the tissue-specific activity of the enhancer. To demonstrate the specificity of the enhancer in vivo, we examined the in vivo interaction of factors with the MLDE in T cells, immature macrophage cells, and in macrophage cells. Although identical DNase I protection activity is present in extracts from all tested cell lines in vitro, the in vivo interaction of proteins is restricted to mature macrophage cells. The presence of factors capable of interacting with the enhancer is not sufficient for enhancer activity, suggesting that the process of differentiation results in factor accessibility for the MLDE. Analysis of the MLDE methylation state revealed a correlation between demethylation of the single CpG dinucleotide within the MLDE sequence and the in vivo interaction of proteins.