Analysis of the Ifng locus

How is the expression of the signature T helper 1 (T_H1)-cell-associated cytokine interferon-γ (IFNγ) regulated at the level of chromatin in CD4⁺ T_H cells? Two papers published in Nature Immunology examined different aspects of the Ifng locus and identified multiple distal regulatory elements that modulate the expression of this gene and specific epigenetic marks that are associated with distinct T_H-cell subsets.

Little is known about the Ifng locus compared with the locus encoding the T_H2-cell-associated cytokines interleukin-4 (IL-4), IL-5 and IL-13, so using computational, chromatin-based and functional approaches Schoenborn et al. searched for potential regulatory elements of the Ifng locus. They identified eight conserved non-coding sequences (CNSs) within 57 kilobases upstream or downstream of the gene, which, owing to their sequence conservation across mice, rats and humans, identified them as potential regulatory elements.

These authors also used another method to distinguish regulatory sites by measuring their sensitivity to cleavage by DNase I. Several DNase-I-hypersensitivity sites were identified in the Ifng locus in T_H1 and T_H2 cells, and the sites were overlapping or adjacent to CNSs in these subsets, respectively. Interestingly, the set of DNase-hypersensitivity sites in T_H1 cells was almost completely distinct from those identified in T_H2 cells, indicating that distinct distal regulatory elements might function in different T_H-cell subsets. In addition, distinct patterns of CpG methylation at the CNSs were observed between the two T_H-cell subsets. Further analysis showed that a subset of these regulatory elements enhanced gene expression, whereas others acted mainly as boundary elements, which are barriers that protect the gene from other regulatory elements or flanking sequences associated with neighbouring genes. Schoenborn et al. also examined histone modifications in the Ifng locus and found a distinct pattern of methylation of histone 3 at lysine 4 (H3-K4) and H3-K27, with enrichment of H3-K4 methylation in T_H1 cells and increased H3-K27 methylation in T_H2 cells.

Chang and Aune focused on histone-methylation marks across the Ifng locus and asked whether there were differences in the methylation of H3-K9 and H3-K27 in T_H2 cells compared with T_H1 cells. They focused on H3-K9 and H3-K27, because methylation of these sites is mainly associated with gene silencing and it is known that Ifng expression is silenced during T_H2-cell development. They found that H3-K9-methylation marks were detected across the Ifng locus early after T-cell activation. However, differentiated T_H2 cells selectively repressed H3-K9 methylation and gained H3-K27-methylation marks. By contrast, methylation marks at H3-K27 were not detected in T_H1 cells, whereas H3-K9 methylation was sustained. These data indicate that methylation of H3-K9 and H3-K27 is dynamic at the Ifng locus. Further analysis showed that both of the T_H2-associated transcription factors signal transducer and activator of transcription 6 (STAT6) and GATA3 extinguished H3-K9-methylation marks and increased H3-K27-methylation marks across the Ifng locus in T_H2 cells, creating an unfavourable environment for transcription of the gene.

So, taken together the data show that Ifng expression is regulated by multiple regulatory elements within the Ifng locus and identify dynamic epigenetic marks that are distinct in CD4⁺ T_H-cell subsets. Defining which transcription factors bind to these regulatory sites and the impact of histone methylation on transcriptional regulation of Ifng will provide further insights into the mechanistic events that lead to gene regulation in T-cell differentiation.