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Remodeling of Il4-Il13-Il5 locus underlies selective gene expression

Nature Immunology volume 25, pages 2220–2233 (2024)Cite this article

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Abstract

The type 2 cytokines, interleukin (IL)-4, IL-13 and IL-5 reside within a multigene cluster. Both innate (ILC2) and adaptive T helper 2 (TH2) lymphocytes secrete type 2 cytokines with diverse production spectra. Using transcription factor footprint and chromatin accessibility, we systemically cataloged regulatory elements (REs) denoted as SHS-I/II, KHS-I/II, +6.5kbIl13, 5HS-I(a, b, c, d, e), 5HS-II and 5HS-III(a, b, c) across the extended Il4-Il13-Il5 locus in mice. Physical proximities among REs were coordinately remodeled in three-dimensional space after cell activation, leading to divergent compartmentalization of Il4, Il13 and Il5 with varied combinations of REs. Deletions of REs revealed no single RE solely accounted for selective regulation of a given cytokine in vivo. Instead, individual RE differentially contribute to proper genomic positioning of REs and target genes. RE deletions resulted in context-dependent dysregulation of cytokine expression and immune response in tissue. Thus, signal-dependent remodeling of three-dimensional configuration underlies divergent cytokine outputs from the type 2 loci.

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Fig. 1: Genome-wide transcription, TF loading and 3D conformation in ILC2s and TH2 cells before and after activation.
Fig. 2: Activation induced TF loading and inferred CTCF loops across the type 2 cytokine locus.
Fig. 3: Activation induced 3D remodeling of type 2 cytokine loci in ILC2 and TH2 cells.
Fig. 4: Contribution of enhancers and structural REs during allergic lung inflammation.
Fig. 5: REs downstream of Il5 differently contribute to chromatin architecture.
Fig. 6: Deletion of +6.5kbIl13 alters TAD structure, enhancer accessibility and IL-13 and IL-5 expression of ILC2s.
Fig. 7: Conserved REs and organization of the IL4-IL13-IL5 locus in human TH2 cells.

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Data availability

Raw and processed data for ATAC-seq, mRNA-seq, ChIP-seq and Hi-C generated in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GSE266349). Raw and processed data for ATAC-seq and mRNA-seq of mouse ILC2s were obtained from our previously published study (GSE131996)14. Source data are provided with this paper.

Code availability

No custom-made code was used in the analysis except for ATAC-seq analysis pipeline (available on request). The pipelines for analysis can be obtained by e-mailing hiroyuki.nagashima@nih.gov.

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Acknowledgements

We thank H. Y. Shih for critical input in interpreting and analyzing the data. We thank S. Dell’Orso and F. Naz (Genome Analysis Core Facility, NIAMS); J. Simone, J. Lay and K. Tinsley (Flow Cytometry Section, NIAMS); S. Jung, H. W. Sun, A. Uhlman, V. Chaitankar and S. R. Brooks (Biodata Mining and Discovery Section, NIAMS); and C. Liu (Transgenic Core, NHLBI) for their technical support. This study used the high-performance computational capabilities of the Biowulf Linux cluster at the NIH. This work was supported by the Intramural Research Programs of NIAMS (grant no. ZIA AR041159 to J.J.O’S.). H.N. was supported by the JSPS Research Fellowship for Japanese Biomedical and Behavioural Researchers at NIH (grant no. 71703).

Author information

Author notes

  1. Franziska Petermann

    Present address: NGS Core Facility, German Cancer Research Center, Heidelberg, Germany

Authors and Affiliations

  1. Lymphocyte Cell Biology Section, NIAMS, NIH, Bethesda, MD, USA

    Hiroyuki Nagashima, Justin Shayne, Franziska Petermann, Yuka Kanno & John J. O’Shea

  2. Biodata Mining and Discovery Section, NIAMS, NIH, Bethesda, MD, USA

    Kan Jiang

  3. Genomics and Immunity Section, NIAMS, NIH, Bethesda, MD, USA

    Aleksandra Pękowska

  4. Dioscuri Centre for Chromatin Biology and Epigenomics, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

    Aleksandra Pękowska

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Contributions

H.N. and J.J.O’S. initiated and designed the project. H.N. performed experiments, analyzed, visualized the data and drafted the manuscript. J.S. performed and analyzed experiments. K.J. contributed the computational analysis. F.P. assisted with the in situ Hi-C. A.P. contributed the design of focused Hi-C. Y.K. and J.J.O’S. supervised the project and edited the manuscript.

Corresponding authors

Correspondence to Hiroyuki Nagashima or John J. O’Shea.

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The authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Gene expression, TF loading, and in situ Hi-C of ILC2s and Th2 cells, related to Fig. 1.

a and b, Gene expression of transcription factors in mouse Th2 and ILC2s by mRNA-seq (GSE131996). c, Aggregated NFAT1 load near HIGs (+/-50kb from TSS) were calculated and binned into 3 categories (common (n = 45), ILC2 specific (n = 70) or Th2 specific (n = 49)) and graphed (Y-axis) for 4 sample conditions (X-axis), as in Fig. 1d. The lower and the upper edge of the box represent the first percentile and the third percentile of the data, respectively. The line inside the box represents the median. The whiskers represent the minimum, maximum and its variability in comparison to the interquartile range. A two-sided t test was used to statistically evaluate the difference of the NFAT1 load between ILC2 specific HIGs and Th2 specific HIGs. d, Interaction frequency (Y axis) vs distance (X axis) of chromatin, measured by HiC for 8 samples. e, Percentage of local chromatin interactions. f, Percentage of interchromosomal interactions. g, Number of TADs detected in individual samples. All TADs are merged by merge2Dbed.pl function by HOMER for downstream analysis.

Source data

Extended Data Fig. 2 Genomic mapping of REs controlling type 2 cytokine locus, related to Fig. 2.

a and b, Five select regions for SHS-I/II, KHS-I/II, +6.5kbIl13, 5HS-II/5HS-III (a + b + c), and 5HS-I(a+b+c+d+e) identified in Fig. 2 were further magnified with phyloP Scores (Mammal Basewise Conservation of 59 vertebrate) showing DNA conservation across species (red; highly conserved, blue; less conserved) and CTCF, GATA3, and ATAC peaks in stimulated ILC2s. The location and directionality of CTCF motifs are depicted with red arrows indicating forward and blue indicating reverse in a. GATA3 motifs are depicted in purple rectangles in a (1 motif) and b (5 motifs). Data from two biological replicates were used for presentation.

Extended Data Fig. 3 Remodeling of Il13/Rad50/Il5 loci induced by SDTFs.

A compiled view of Il13/Rad50/Il5 loci showing chromatin accessibility, TFs binding and HiC interaction in ILC2s stimulated with IL-33 plus NMU. Data represents chr11: 53,623,000-53,745,000 (122kB). Red circles illustrate interactions between enhancers proximity to Il13 and Il5 and the locus control region (LCR).

Extended Data Fig. 4 Gating strategy for lung cells, related to Fig. 4.

a and b, A schematic gating strategy for flow cytometry analyses for mouse lung cells.

Extended Data Fig. 5 Humoral immune response of 5HS-Ie, 5HS-II and 5HS-III(a+b+c) KO mice, related to Fig. 4.

Quantification of papain−specific IgM, IgG1 and IgE in WT (naïve; n = 8, papain; n = 24), 5HS-Ie KO (naïve; n = 4, papain; n = 8), 5HS-II KO (naïve; n = 4, papain; n = 11) and 5HS-III(a+b+c) KO (naïve; n = 5, papain; n = 16) mice before and after administration of Papain, as in Fig. 4. Graphs show mean ± SEM.

Source data

Extended Data Fig. 6 Chromatin architecture of 5HS-II and 5HS-III(a+b+c) KO ILC2s, related to Fig. 5.

A split view of HiC heatmap of WT, 5HS-II KO and 5HS-III(a+b+c) KO ILC2s with or without stimulation with IL-33 + NMU for 1 h, as in Fig. 5c.

Extended Data Fig. 7 Contribution of +6.5kbIl13 during chronic lung inflammation, related to Fig. 6.

a, mRNA expression of Il4, Il13 and Il5 in WT and +6.5kbIl13 KO ILC2s stimulated with IL-33 + NMU measured by qPCR. The data were normalized to Actb, then further normalized to non-stimulated WT cells ( = 1). Graphs are representative plots from three independent experiments, and triplicates summary are shown. b-d, WT (n = 13) and +6.5kbIl13−KO (Δ+6.5kbIl13, n = 10) mice were challenged with papain to induce lung inflammation. c, Flow cytometry for infiltrating cells recovered from the lungs. Cells evaluated were total live cells, eosinophils (CD11b+ Siglec-F+), neutrophils (CD11b+ Gr1+), Th2 (CD3ε+ TCRβ+ CD4+ CD44+ Foxp3- GATA3+) and ILC2s (Lin− Thy1+ CD127+ GATA3+). d, Cytokine production from Th2 cells was evaluated by flow cytometry measuring IL-4, IL-13 and IL-5. Graphs in c and d show mean ± SEM. Statistical significance is depicted as ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 (Two-sided t test).

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

List of HIGs and WIGs, related to Fig. 1b.

Supplementary Table 2

List of common and cell-type-specific HIGs and WIGs, related to Fig. 1c.

Supplementary Table 3

List of total and differentially regulated TADs, related to Fig. 1e.

Supplementary Table 4

Guide RNAs and primers for the generation of mice.

Supplementary Table 5

Custom oligo pool for focused Hi-C.

Source data

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Source Data Extended Data Fig. 5

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Nagashima, H., Shayne, J., Jiang, K. et al. Remodeling of Il4-Il13-Il5 locus underlies selective gene expression. Nat Immunol 25, 2220–2233 (2024). https://doi.org/10.1038/s41590-024-02007-4

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