The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulatedin vivoby an inducible tissue-specific enhancer

Abstract

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ؎ 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P ‫؍‬ 0.004) and more variable level of activity (29% ؎ 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

T Cell-Specific Expression of the Human TNF-α Gene Involves a Functional and Highly Conserved Chromatin Signature in Intron 3

“…Moreover, these DH sites, which can be detected by sensitivity to DNase I, have frequently provided the first evidence of the existence of distant regulatory elements located even several kilobases upstream or downstream of genes, or in some cases within introns (15)(16)(17)(18)(19)(20)(21).…”

The Human IL-3 Locus Is Regulated Cooperatively by Two NFAT-Dependent Enhancers That Have Distinct Tissue-Specific Activities

“…The Ϫ14-kb IL-3 enhancer functions exclusively in T cells, and this specificity is mediated by a composite NFAT/Oct element that recruits NFAT and Oct factors together with the NFAT cofactor NIP45 and the lymphoid-specific Oct cofactor OCA-B (10). In contrast, the Ϫ3-kb GM-CSF enhancer functions in a much wider range of NFAT-expressing cells such as T cells, myeloid cells, and endothelial cells (12), and is activated via composite NFAT/AP-1 elements (13). The fact that the locus has evolved with two distinct enhancers also makes it more likely that the IL-3 and GM-CSF genes are independently regulated.…”

Partners in transcription: NFAT and AP-1

“…The cell-type speci®city of GM-CSF expression di ers from that of IL-3: GM-CSF is expressed by lymphocytes as well as myeloid cells, while IL-3 is exclusively expressed by lymphoid cells. This di erence has been attributed to the fact that IL-3 gene expression appears to be controlled by a separate upstream enhancer, that contains NFAT: Oct elements rather than NFAT:AP-1 composite sites (Bert et al, 2000;Cockerill et al, 1999;Duncli e et al, 1997). However the use of a mutant NFAT1 protein incapable of AP-1 interaction shows clearly that both IL-3 promoter activity and expression of the endogenous IL-3 gene required cooperative interactions between NFAT and AP-1, thus placing IL-3 ®rmly in the category of NFAT:AP-1-dependent genes (Macia n et al, 2000).…”