ABSTRACT
The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Genes, fms/genetics , Introns/genetics , Macrophages/metabolism , Promoter Regions, Genetic/genetics , Receptor, Macrophage Colony-Stimulating Factor/biosynthesis , 3T3 Cells/metabolism , Animals , Base Sequence , Cell Line , Deoxyribonuclease I/metabolism , Gene Expression Profiling , Genes, Reporter , Humans , Luciferases/biosynthesis , Luciferases/genetics , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides, Antisense/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription Factors/metabolism , TransfectionABSTRACT
Heteroplasmic populations of mtDNA, consisting of normal mtDNA and mtDNA with large deletions, are found in the skeletal muscle and other tissues of certain patients with mitochondrial respiratory chain deficiencies, particularly in those with the CPEO (chronic progressive external ophthalmoplegia) phenotype. To study the developmental genetics of this mitochondrial disorder, the distribution of the deleted mtDNA in a wide range of tissues of different embryonic origins (total 34 samples from 27 tissues obtained at autopsy) was investigated in a patient with the CPEO syndrome. Three species of partially deleted mtDNA were observed, with deletions of 2.3 kb, 5.0 kb and 6.4 kb. Their tissue distribution suggests that the mtDNA deletions have occurred very early during embryonic development, prior to the differentiation events that lead to the formation of the three primary embryonic germ layers, and that the partially deleted mtDNA species were segregated during development mainly to the skeletal muscle and to tissues of the central nervous system.
