Nase I HS and Genomic Requirements Transgenics

Concentrated work in recent years has offered a more complete picture of the genomic requirements for cell- and signal-specific regulation of IFN-y (Fig. 1). Transgenic mouse models of IFN-y regulation suggest that regions distal to the ifng gene are required to confer normal IFN-y expression profiles (Soutto et al. 2002; Young et al. 1989). An 8.6-kb fragment of human genomic DNA containing the full-length ifng gene and 2.7 kb of upstream sequence fails to provide tissue-specific IFN-y expression. Meanwhile, a bacterial artificial chromosome (BAC) transgene containing a 191-kb fragment of genomic DNA surrounding the ifng gene recapitulates cell-specific and optimal signal-specific IFN-y expression patterns, thereby indicating a requirement for distal regulatory sites (Soutto et al. 2002).

In this context, several putative regulatory regions have been identified in and around the ifng gene based on DNase I HS analysis. Three HS sites are located in introns (HS I, II, and III), with two of these regions preferentially appearing in T helper 1 (Th1) cells (Agarwal and Rao 1998; Hardy et al. 1987). These sites fall within the previously mentioned 8.6-kb genomic fragment that

Ifng gene structure and putative genomic regulatory regions

Ifng gene structure and putative genomic regulatory regions

HS I HS II HS III

M = DNA methylation site A = Histone acetylation site 1 = 1st Exon

* = Region has enhancer activity

Fig. 1 Epigenetic structure of the human ifng locus on chromosome 12. Exons are represented by gray boxes with exon 1 indicated. HS, DNaseI hypersensitivity site; CNS, conserved noncoding sequence. Known sites of DNA methylation and histone acetylation are indicated. See text for associated references is not completely sufficient to support appropriate IFN-y expression. Recent studies have characterized additional regulatory sites that are located outside the 8.6-kb fragment, which have begun to provide some insight into the cooperative effects of distal and proximal regulatory elements in controlling IFN-y gene expression profiles.

Early efforts to identify regulatory regions in the ifng gene were hampered by insufficient sequence information beyond the proximal portions of the ifng gene. However, with the completion of the human, mouse, rat, and other species' genomes, comparative sequence analysis is now possible on a larger scale and has already been proven as a powerful analytical tool to identify genomic regions of homology that are likely to harbor regulatory sequences (Gu and Su 2005; Nardone et al. 2004). For instance, utilizing DNA sequence comparisons between species as a tool to identify conserved sequence motifs in the ifng gene (generally greater than 100 bp in length with approximately 70% sequence identity; Dermitzakis et al. 2005), Lee and colleagues have found another HS site that is approximately 5 kb upstream of the ifng gene (5'CNS) (Dermitzakis et al. 2005; Lee et al. 2004). Interestingly, these analyses also revealed that previously identified HS sites in the ifng intronic regions are situated within or in close proximity to conserved noncoding sequences (CNS). Taking a similar approach, Shnyreva et al. have confirmed the 5'CNS and identified a conserved site 18 kb downstream of the mouse ifng gene (CNS2) that has Thl-specific HS activity (Shnyreva et al. 2004). Our group has identified another HS 5' to the ifng gene, located-approximately 3.5 kb upstream, that is responsive to cytokine stimulation in human NK cells (Bream et al. 2004). Although this region does not map to a CNS site, we did identify a conserved Stat5 binding motif within the HS region that is near the same genomic position upstream of both the mouse and human ifng genes. In a more recent publication, Hatton et al. reported on additional CNS sites at -22 kb, -34 kb and -55 kb. They found that the CNS at -22 kb also contained a number of transcription factor binding sites, was found to bind T-bet, and enhanced promoter activity. When this CNS site was deleted using a transgenic approach, it eliminated CD4+/CD8+ T cell and NK cell IFN-y expression in response to T cell receptor signaling or interleukin signaling, respectively (IL-12 + IL-18) (Hatton et al. 2006).

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