Unexpected Characteristics of the IFN-γ Reporters in Nontransformed T Cells

Abstract

Analysis of the IFN-γ promoter has primarily been conducted by transient expression of reporter constructs in transformed cells. However, the activity of cis elements may differ when expressed transiently compared with their activity within native chromatin. Furthermore, the transcription factors and signaling mechanisms in transformed cells may differ from those in normal T cells. To analyze IFN-γ promoter regulation in normal T cells, we developed a novel retroviral bottom-strand reporter system to allow the chromatin integration of promoter regions in primary developing T cells. As controls, both the IL-2 and IL-4 promoters were inducible in this system, with the IL-4 reporter having Th2-specific activity. Strikingly, the IFN-γ promoter exhibited constitutive activity in both Th1 and Th2 subsets, in contrast to the behavior of the endogenous IFN-γ gene, which is inducible only in Th1 cells. In mapping this activity, we found that the AP-1/GM-CSF site in the distal promoter element is the most critical element for the constitutive activity. Transgenic reporter lines for the IFN-γ promoter confirmed the constitutive behavior of the isolated IFN-γ promoter. This constitutive activity was resistant to inhibition by cyclosporin A and was independent of Stat4 and p38 mitogen-activated protein kinase. These results suggest that IFN-γ promoter regulation may require cis elements residing either downstream or >3.4 kb upstream of the transcriptional start site, involving repression of constitutive activity.

Friend of GATA Is Expressed in Naive Th Cells and Functions As a Repressor of GATA-3-Mediated Th2 Cell Development

“…Our reporter analyses indicated that GATA-3 transactivates all of the IL-5, IL-4, and IFN-␥ promoters in Jurkat cells (15,51), differently from EL-4 cells or Th2 cells (11,13). The short fragment of the IFN-␥ promoter does not behave in parallel with the endogenous IFN-␥ gene (52), and there is also evidence that the expression of IFN-␥ gene is regulated at the chromatin accessibility (21), which cannot be reflected by reporter assays. Our reporter assays revealed that FOG represses the GATA-3-mediated transactivation of all the cytokine promoters, including IFN-␥.…”

A Distal Region in the Interferon-γ Gene Is a Site of Epigenetic Remodeling and Transcriptional Regulation by Interleukin-2

“…Interestingly, this methylation site is evolutionarily conserved, because it is present in other vertebrates with the exception of avians (47). Regardless, the proximal promoter regions important for TCRinduced activation are not sufficient for normal tissue-specific expression or cytokine-induced IFNG gene induction (8,9).…”

“…Thus, in vivo control of IFN-␥ transcription is likely to involve the cooperative effects of regions both proximal and distal to the IFNG gene (8). Accordingly, the 5Ј-flanking region of the IFNG gene has been suspected of harboring crucial regulatory elements (9), and a PHA/PMA-inducible HS site estimated to be ϳ3 kb upstream was found in Jurkat T cells in 1987 (28). A recent paper by Lee and colleagues (60) describes a distal Th1-specific HS site that was estimated to be ϳ5 kb upstream of the murine IFNG gene.…”

“…In addition, transgenic mice that contain the full-length human IFNG gene and ϳ2.7 kb of upstream sequence have similar signal-specific regulatory patterns to the endogenous gene, yet the responses are relatively weak (8). Therefore, it is likely that regions more distal from the core IFNG promoter are involved in determining tissue-specific and optimal signalspecific expression of IFN-␥ (9).…”

Cutting Edge: Identification of an Alternative GATA-3 Promoter Directing Tissue-Specific Gene Expression in Mouse and Human

“…DO11.10 ␣␤ TCR-transgenic mice (9,20), murine rIL-4, and rIL-12 (21) have been described. Anti-CD28 (37.51) and CD3 (500A2) were gifts from Dr. J. P. Allison (Berkeley, CA) and were purified by affinity chromatography from culture supernatant or used as ascites.…”