Shaping Il4 gene expression

Transcription of the T helper 2 (T_H2)-associated cytokine genes — interleukin-4 (IL4), IL5 and IL13 — is controlled by the T_H2 cell master regulator GATA-binding protein 3 (GATA3). However, the molecular basis of GATA3-mediated gene regulation during T_H2 cell development is unclear and controversial. Tanaka et al. now show that binding of GATA3 to DNase I hypersensitive site 2 (HS2) in the second intron of the Il4 locus is specifically required for chromosomal modifications at this locus that allow transcription of Il4.

Numerous regulatory elements in the T_H2 cytokine locus have been identified, but whether T_H2-associated cytokine expression is controlled by a single element or by the coordinated activity of multiple elements is not known. To address this issue, the authors generated a series of mutant mice that lack each hypersensitive element in the Il4–Il13 locus and assessed the effect of each deletion on cytokine production. T_H2 cells from mice that lack HS2 produced the lowest levels of IL-4 following activation, whereas the expression of other T_H2-type cytokines by these cells was similar to wild-type T_H2 cells. These data suggest a specific role for HS2 in IL-4 expression. Deletion of other regulatory elements also impaired IL-4 expression, but to a lesser extent, suggesting that multiple elements are required for complete lineage-specific expression of IL-4. By contrast, naive T cells that lack the conserved GATA3-response element (GCRE) in the Il13 locus gave rise to wild-type numbers of IL-4-producing T cells but few IL-13-producing T cells in T_H2 cell-inducing conditions, indicating that this element regulates Il13 transcription.

Next, the authors assessed whether GATA3 is linked to the function of the HS2 enhancer. Unlike in wild-type T_H1 cells, overexpression of GATA3 in HS2-deficient T_H1 cells did not result in IL-4 expression. Furthermore, GATA3 directly binds to HS2 during T_H2 cell differentiation, as determined by chromatin immunoprecipitation analysis.

GATA3 functions mainly as an epigenetic modifier, so it is possible that binding of GATA3 to HS2 is required for transcription-permissive epigenetic changes at the Il4 locus. Indeed, acetylation of histone H3 at Lys9 and Lys14, and trimethylation of histone H3 at Lys4 (all of which are permissive modifications) were impaired in HS2-deficient T_H2 cells, but only at the Il4 locus. By contrast, deletion of GCRE resulted in impaired methylation of histone H3 at Lys4 at the Il13 locus but not the Il4 locus.

Finally, antigen-specific IgG1 and IgE levels, eosinophilia and airway hyperresponsiveness to acetylcholine were reduced in HS2-deficient mice compared with wild-type mice in models of allergic lung inflammation, confirming that the T_H2 cell response was impaired in HS2-deficient mice.

So, HS2 is a crucial GATA3-binding site in the Il4 locus and is required for the GATA3-mediated epigenetic modifications that are necessary for lineage-specific IL-4 expression.