Chromatin landscape dynamics of the Il4-Il13 locus during T helper 1 and 2 development

Abstract

Il4 and Il13 encode the canonical T helper 2 (TH2) cytokines responsible both for promoting immune responses against extracellular pathogens and, when misregulated, causing allergic and autoimmune disease. The expression potential of these genes undergoes developmentally programmed repression and enhancement during commitment of naïve CD4+ T cells to the mature T helper 1 (TH1) and TH2 fates, respectively. Thus, like the globin locus, the TH2 cytokine locus provides a highly tractable system to study a developmental fate choice leading to alternative transcriptional states of either silence or permissivity. We used quantitative chromatin immunoprecipitation and RT-PCR to correlate changes in the transcriptional states of Il4 and Il13 with markers of permissive chromatin across the Il4-Il13 locus in naïve CD4+ T cells undergoing TH1 and TH2 differentiation. We provide evidence that DNaseI hypersensitive site V in the Il4 3' enhancer is the likely target for signals maintaining Il4 and Il13 transcriptional permissivity in naïve cells. We also demonstrate rapid acquisition of differences in H3 acetylation between TH1- and TH2-primed cells, indicating a developmentally early role for cytokine signaling in the process of TH cell fate determination. Finally, we show that transcriptional repression correlates with the disappearance of permissive H3 modifications from everywhere in the Il4-Il13 locus except hypersensitive site IV, suggesting a critical role for this element in the maintenance of transcriptional repression. Our findings are consistent with a progressive regulatory element activation/deactivation model of TH1/TH2 development.

Fig. 2.

Permissive histone H3 modification at the Il4–Il13 locus during T_H1 development. Shown are ChIP plots depicting fold-enrichment for AcK9/14 (circles) and 2MeK4 (squares) across the Il4–Il13 locus in CD4⁺CD62L^Hi naïve T cells (A), 48-h T_H1-primed CD4⁺ T cells (B), and resting A.E7 cells (C). Error bars are SEM of n ≥ 2 except for the following where n = 1: naïve (AcK9/14, sites F, G, H and P; 2MeK4, sites A, E′, and J) and A.E7 (AcK9/14, sites B, C E′, F, I, and L; 2MeK4, sites E′, I, and L). 2MeK4 data are not shown for 48-h-primed cells. At the top of the figure is a physical map of the chromosome 11 Kif3a–Il4–Il13–Rad50 locus. Horizontal black arrows represent complete (solid lines) or partial (dotted lines) transcription units. Tall black rectangles represent exons. Arrowheads indicate the location of T_H2-specific/constitutive (filled symbols) and T_H2-specific/activation-dependent (open symbols) DNase I HSs. The small black rectangle represents a region of high sequence conservation between mouse and human (CNS1). Diamond-encased letters (A–P) represent the names and locations of PCR primer pairs. Dotted vertical lines in each plot bracket the locations of transcription units.

Fig. 1.

Kif3a, Il4, Il13, and Rad50 mRNA expression in developing T_H1 and T_H2 cells. CD4⁺CD62L^Hi naïve T cells (A and B), CD4 T cells primed for 48 h in T_H1(A) or T_H2(B) skewing condition, the T_H1 clone A.E7 (A), and the T_H2 clone D10.G4 (B) were harvested for RNA after 4 h of activation with PMA and ionomycin. Relative expression of Kif3a, Il4, Il13, and Rad50 was determined by quantitative real-time PCR. The ratio of Hprt-normalized expression in T cells and NIH3T3 cells is depicted in arbitrary units (AU). Error bars are SEM of n ≥ 2.

Fig. 5.

Distribution of serine 10 phosphorylation on histone H3 in A.E7, D10.G4, and NIH3T3 cells. Shown are ChIP plots depicting fold-enrichment of PhS10 across the Il4–Il13 locus of resting A.E7 (circles), D10.G4 (squares), and NIH3T3 (triangles) cells. Error bars are SEM of n ≥ 3. The top of the figure is as described for Fig. 2.

Fig. 3.

Histone H3 modification at the Il4–Il13 locus during T_H2 development. Shown are ChIP plots depicting fold-enrichment for AcK9/14 (circles) and 2MeK4 (squares) across the Il4–Il13 locus in CD4⁺CD62L^Hi naïve T cells (A), 48-h T_H1-primed CD4⁺ T cells (B), and resting D10.G4 cells (C). Error bars are SEM of n ≥ 2 except for the following where n = 1: naïve (AcK9/14, sites F–H and P; 2MeK4, sites A, E′, and J). In C, the AcK9/14 enrichment for probe G is 150 ± 30. 2MeK4 data are not shown for 48-h-primed cells. The top of the figure is as described for Fig. 2. The naïve plot (A) is the same as shown in Fig. 2 A.

Fig. 4.

Effect of activation on H3 modification at the Il4–Il13 locus of a T_H2 clone. Shown are ChIP plots depicting fold-enrichment for AcK9/14 (Upper) and 2MeK4 (Lower) across the Il4–Il13 locus of D10.G4 resting (filled squares) or after 4-h culture with PMA/ionomycin (open squares). Error bars are SEM of n = 2 for activated and n ≥ 3 for resting. Resting data are from Fig. 3C. The top of the figure is as described for Fig. 2.