Antigen and cytokine receptor signals act in synergy to direct the differentiation of CD4+ T cells. These signals initiate reciprocal activation and silencing of the interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) cytokine gene loci, changes that are heritably maintained in the resulting T helper type 1 (T(H)1) or T(H)2 cells and their progeny. Early, unpolarized transcription and chromatin remodeling of the poised cytokine genes of naive T cells is followed by consolidation and spreading of epigenetic changes and the establishment of self-reinforcing transcription factor networks. Recent studies have begun to elucidate the molecular mechanisms that establish and maintain polarized cytokine gene expression, and thus the cellular identity of differentiated helper T cells.

The relation of CpG methylation to gene silencing is well established, but the contribution of DNA demethylation to gene expression during cell differentiation remains unclear. We show that the IL-4 locus undergoes a complex series of methylation and demethylation steps during T helper cell differentiation. The 5' region of the IL-4 locus is hypermethylated in naive T cells and becomes specifically demethylated in Th2 cells, whereas a highly conserved DNase I-hypersensitive region at the 3' end shows the converse behavior, being hypomethylated in naive T cells and becoming methylated during Th1 differentiation. 5' demethylation is not required for chromatin remodeling or primary transcription of the IL-4 gene but is strongly associated with efficient, high-level induction of IL-4 transcripts by differentiated Th2 cells.

Large-scale cross-species DNA sequence comparison has become a powerful tool to identify conserved cisregulatory modules of genes. However, bioinformatic analysis alone cannot reveal how an evolutionarily conserved region regulates gene expression: whether it functions as an enhancer, silencer, or insulator; whether its function is cell-type restricted; and whether biologically relevant transcription factors bind to the element. Here we combine bioinformatics with wet-lab techniques to illustrate a general and systematic method of identifying functional conserved regulatory regions of genes. We applied this approach to the interferongamma (IFN-␥) gene. Comparison of human and mouse IFN-␥ reveals a highly conserved non-coding sequence located ϳ5 kb 5 of the transcription start site. This region coincides with constitutive and inducible DNase I hypersensitivity sites present in IFN-␥-producing Th1 cells but not in Th2 cells that do not produce IFN-␥. Histone methylation at the 5 conserved non-coding sequences indicates a more accessible chromatin structure in Th1 cells compared with Th2 cells. This element binds two transcription factors known to be essential for IFN-␥ expression: nuclear factor of activated T cells, an inducible transcription factor, and T-box protein expressed in T cells, a cell lineage-restricted transcription factor. Together, these findings identify a highly conserved distal enhancer in the IFN-␥ cytokine locus and validate our approach as a successful method to detect cis-regulatory elements.Gene transcription is regulated by the binding of transacting factors to short DNA elements (5-8 bp) contained within larger blocks of cis-regulatory sequences (100 -400 bp) (1). An important cis-regulatory module is located at the proximal promoter, which specifies the site of transcriptional initiation. In metazoans, however, the levels and cell specificity of gene expression are generally controlled by distal regulatory modules (enhancers and silencers) that may be located in introns or intergenic regions and that potentiate or repress gene transcription. Typically, multiple regulatory modules are associated with a single gene, and different combinations of modules are utilized to control gene expression in different cell types at different developmental stages or under different conditions of stimulation (1). A characteristic feature of the modules is the presence of clustered binding sites for relevant transcription factors. For instance, the promoters and distal enhancers of genes expressed in muscle are enriched in binding sites for muscle-specific transcription factors, including Mef-2, Myf, and SRF (2, 3).Recently, there has been much interest in the possibility of using comparative sequence analysis to identify gene regulatory elements. In mammals, only about 5% of the genome is estimated to encode proteins. The bulk of non-coding DNA is not conserved between species, but long-range sequence comparisons reveal islands of highly conserved non-coding sequences (CNS) 1 in a sea of non-conserved DN...