Analysis of intergenic transcription in the human IL-4/IL-13 gene cluster - PubMed (original) (raw)

Analysis of intergenic transcription in the human IL-4/IL-13 gene cluster

David F Rogan et al. Proc Natl Acad Sci U S A. 2004.

Abstract

During the differentiation of naïve CD4+ precursors to T helper 1 (Th1) or Th2 effector cells, several epigenetic changes occur in a lineage-specific manner at the IFN-gamma or IL-4/IL-13 loci. These changes result in alterations in the chromatin structure of these loci and, hence, lineage-restricted expression of the corresponding cytokines. Intergenic transcripts have recently been shown to regulate the expression of genes in the beta-globin locus; therefore, we have examined the Th2 cytokine gene cluster during human Th1/Th2 differentiation and in a transgenic mouse line containing the human IL-4/IL-13 genes for intergenic transcripts. We show for the first time that intergenic transcription of this locus is restricted to tissues and lineages in which IL-4 and IL-13 are expressed. We also show that intergenic transcription in the IL-4/IL-13 locus is up-regulated after Th2 differentiation. Furthermore, we demonstrate that the Th2 cytokines and intergenic transcripts are detectable in the thymus. We propose that intergenic transcription is tightly associated with transcriptional competence for the Th2 cytokines and may play a role in their regulation. These results support a progressive differentiation model of T cell lineage commitment.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Human IL-4/IL-13 locus. The position and orientation of genes are indicated by arrows, and exons are indicated by gray boxes below arrows. The positions of the known regulatory regions CNS-1 and CNS-2 are indicated. The positions of intergenic transcription amplimers (IG1–5) examined in this study are also shown.

Fig. 2.

Fig. 2.

Th2-specific expression of human IL-4 and IL-13 in transgenic mice. (A) Expression of IL-4 and IL-13 is restricted to lymphoid tissues. RNA was isolated from transgenic lung (Lu), liver (Li), heart (Ht), kidney (Ki), and thymus (Th), reverse-transcribed, and analyzed by PCR for human and murine IL-4 and IL-13. Number of PCR cycles: β-actin, 18; hIL-4, 28; mIL-4, 28; hIL-13, 24; mIL-13, 28. (B) IL-4 and IL-13 are transcribed in activated CD4+ cells. CD4+ cells were purified from human blood and transgenic and wild-type murine spleens and were either activated with phorbol 12,13-dibutyrate and ionomycin for 18 h or left unactivated. RNA was isolated from each cell population, reverse-transcribed, and analyzed by PCR. Number of PCR cycles: β-actin, 18; hIL-4, 32; mIL-4, 32; hIL-13, 32; mIL-13, 32. (C) Th2-specific expression of human IL-4 and IL-13. CD4+ cells were purified from transgenic and nontransgenic spleens and cultured for 7 days under Th1 or Th2 conditions. The cells were then stimulated for 5 h with anti-CD3ε, and the RNA from each population was reverse-transcribed and used for PCR. The linear range of the PCR for each primer pair was determined by using increasing copies of cDNA as template and is shown in the line graphs. The levels of mRNA expression are shown in the histograms below the gels and are expressed as copies of cDNA per μg of RNA used in the reverse transcription reaction. Number of PCR cycles: β-actin, 18; hIL-4, 22; mIL-4, 22; hIL-13, 22; mIL-13, 22; murine IFN-γ, 16.

Fig. 3.

Fig. 3.

Lymphoid-specific intergenic transcription in the human Th2 cytokine cluster. RNA was isolated from transgenic lung (Lu), liver (Li), heart (Ht), kidney (Ki), and thymus (Th), reverse-transcribed, and analyzed by PCR for human intergenic transcripts IG1–5. –RT indicates control reverse transcription without enzyme (lanes 1, 3, 5, 7, 9, and 11). Number of PCR cycles: β-actin, 20; IG1–5, 34.

Fig. 4.

Fig. 4.

Intergenic transcription after Th1 or Th2 differentiation. (A and B) Expression of IL-4 and IL-13 and intergenic transcripts in murine Th1/Th2 cells. CD4+ cells were purified from transgenic spleens and cultured for 7 days under Th1 or Th2 conditions. The cells were then stimulated for 5 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and the RNA from each population was reverse-transcribed and used in PCR for the genic (A) and intergenic (B) transcripts indicated. Number of PCR cycles: β-actin, 18; hIL-4, 22; mIL-4, 24; hIL-13, 22; mIL-13, 24; murine IFN-γ, 16; IG1–5, 34. (C and D) Expression of IL-4 and IL-13 and intergenic transcripts in human Th1/Th2 cells. CD4+ cells were purified from peripheral blood and cultured for 14 days under Th1 or Th2 conditions. The cells were then stimulated for 5 h with PMA and ionomycin, and the RNA from each population was reverse-transcribed and used in PCR for the genic (C) and intergenic (D) transcripts indicated. Number of PCR cycles: β-actin, 22; hIL-4, 28; hIL-13, 22; RAD50, 28; KIF3A, 28; IFN-γ, 18; IG1–5, 34.

Fig. 5.

Fig. 5.

Transcription across the Th2 cytokine cluster in human fibroblasts. RNA was isolated from resting or activated normal skin fibroblasts and subjected to RT-PCR for the Th2 cytokine cluster genic (A) and intergenic (B) transcripts. Where indicated (+Act) cells were activated with IL-1β, tumor necrosis factor α, and IFN-γ for 24 h. Number of PCR cycles: β-actin, 22; hIL-4, 34; hIL-13, 34; RAD50, 27; KIF3A, 27; GM-CSF, 32; IG1–5, 34.

Similar articles

Cited by

References

    1. Glimcher, L. H. & Murphy, K. M. (2000) Genes Dev. 14, 1693–1711. - PubMed
    1. Murphy, K. M. & Reiner, S. L. (2002) Nat. Rev. Immunol. 2, 933–944. - PubMed
    1. Glimcher, L. H. & Singh, H. (1999) Cell 96, 13–23. - PubMed
    1. O'Garra, A. (2000) Curr. Biol. 10, R492–R494. - PubMed
    1. Cousins, D. J., Lee, T. H. & Staynov, D. Z. (2002) J. Immunol. 169, 2498–2506. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources