1. Th2 Lineage Commitment and Efficient IL-4 Production Involves Extended Demethylation of the IL-4 Gene 
Dong U. Lee, Suneet Agarwal, Anjana Rao 
Immunity 
Volume 16, Issue 5, Pages (May 2002)
DOI: /S (02)00314-X

2. Figure 1
Additional Th2-Specific DH Sites in the IL-4/IL-13 Intergenic Region and Differential Methylation of the IL-4 Gene in Th1 and Th2 Cells Assessed by SmaI and HpaII Digests
(A) From top to bottom, the arrows indicate: a fragment appearing only in D5 samples digested with both DNase I and SmaI, which is bounded by DH sites HSS3 and IV (lanes 2–5); the 7 kb SmaI fragment, which is abundant in D10 SmaI digests (lanes 6–10) but very poorly represented in D5 SmaI digests (lanes 1–5); and a cluster of new DH sites (DH sites 0) in the IL-4/IL-13 intergenic region.
(B) Diagram of the murine IL-13/IL-4/KIF3A locus depicting the position of SmaI sites and the approximate location of the constitutive DH sites found in Th1 cells and all Th2-specific DH sites, including the novel intergenic DH sites 0 observed in (A). The span of the ∼18 kb fragment bounded by DH sites HSS3 and IV is shown for D5 as well as the span of the 7 kb fragment produced by SmaI digestion in D10. Also shown are the position of the probe used in (A) and the methylation status of the SmaI sites in the region depicted (M, methylated; D, demethylated).
(C) Methylation analysis of the IL-4 locus in D5 Th1 and D10 Th2 cells. Genomic DNA was restriction digested as indicated and analyzed by Southern blotting using the 5′ IL-4 probe shown in (E). SmaI and HpaII are CpG methylation-sensitive restriction enzymes, and XmaI and MspI are their respective methylation-insensitive isoschizomers.
(D) Demethylation of IL-4 intron 1 during Th2 differentiation. Naive CD4 cells were directly analyzed or were differentiated for 2 or 3 weeks in Th1 or Th2 polarizing conditions as indicated.
(E) Diagram of the IL-4 gene summarizing the data of parts (C) and (D). The top line shows the scale in kilobases (kb), with 0 located at the BamHI site in the IL-4 promoter. The second line shows the IL-4 gene with the 5′ promoter probe used for Southern analysis, IL-4 and KIF3A exons (black boxes), and DNase I-hypersensitive sites (gray ovals with Roman numerals). Arrows indicate the directions of IL-4 and KIF3A gene transcription. The third line shows restriction sites (unlabeled vertical lines) in the 19 kb BamHI fragment (BamHI, B; SmaI/XmaI, S/X; HpaII/MspI, unlabeled vertical lines). The remainder of the figure is aligned with the locus diagram and explains the fragments observed in the Southern blots of (C) and (D). Uppercase letters D and M indicate complete demethylation or complete methylation of a site as judged by Southern blotting, while lowercase letters indicate partial methylation or demethylation.
Immunity  , DOI: ( /S (02)00314-X)

3. Figure 2
Changes in DNase I Hypersensitivity Occur Independently of Intronic Demethylation
Naive CD4+ T cells were differentiated under Th2 conditions for 8 weeks and subject to DNase I treatment. Genomic DNA was digested with BamHI, and 75% of the purified product was further digested with SmaI. “BamHI only” (panel 1) and “BamHI + SmaI” (panel 2) samples were analyzed by Southern blotting using the 5′ IL-4 probe. In panel 2, the lower arrow indicates the 1.2 kb fragment produced by digestion of the demethylated alleles in the Th2 population, while the upper arrow indicates the intact genomic fragment that is methylated and thus resistant to SmaI digestion. Chromatin remodeling is apparent on methylated alleles as shown by equivalent DNase I hypersensitivity of methylated and demethylated alleles of the IL-4 gene.
Immunity  , DOI: ( /S (02)00314-X)

4. Figure 3
Differential Methylation of the IL-4 Gene in Th1 and Th2 Cells Assessed by McrBC Digestion
Genomic DNA from the indicated cell populations was digested with BamHI + McrBC and analyzed by Southern blot using the 5′ IL-4 probe shown in (D). McrBC cleaves only methylated DNA. For the experiments shown in (B) and for (C), the DNA samples were analyzed on a single gel in each case, but the lanes are shown separated (and in some cases, rearranged) for convenience.
(A) Methylation analysis of the IL-4 locus in D5 Th1 and D10 Th2 cells. TSS, transcription start site (beginning of exon 1).
(B and C) Changes in methylation status of the IL-4 gene during Th differentiation. Purified naive CD4+ T cells or bulk CD4+ T cells from STAT6−/− mice or C57BL/6 controls, and Maf−/− mice or their “wild-type” Maf+/+ littermates were analyzed directly or after differentiation under Th1, Th2, or Th0 (no polarizing cytokines plus added neutralizing antibodies) conditions for 1–8 weeks as indicated. The fragments obtained by McrBC cleavage at the transcription start site (TSS) and exon 2 are indicated.
(D) Diagram of the IL-4 gene summarizing the data of (A)–(C). For details, see Figure 1E.
Immunity  , DOI: ( /S (02)00314-X)

5. Figure 4
PCR Assay Showing Hypomethylation of the IL-4 Promoter in Both Th1 and Th2 Cells
(A) Diagram indicating the locations of PCR primers (arrows) spanning the promoter and exon 1/intron 1/exon 2 regions of the IL-4 gene. Exons 1 and 2 are shown as light gray boxes, purine CpG sequences (which are recognized by McrBC if hemimethylated or symmetrically methylated on cytosines) are shown as dark gray circles, and BamHI and SmaI/XmaI sites are shown as vertical lines. An arrow shows the location of the TATA box. The numbers indicate distance in base pairs from the promoter BamHI site.
(B) Experiment to show linear range of the PCR assay. BamHI-digested DNA was serially diluted 2-fold and subject to PCR. Intensity of the PCR bands was determined by phosphorimager and graphed.
(C and D) Experiment to compare efficiency with which McrBC cleaves in the promoter with widely spaced recognition sites relative to the closely spaced recognition sites in exon 1. A plasmid spanning the IL-4 promoter/exon 1 region was CpG methylated with SssI methylase in vitro. Untreated or methylated plasmid was subjected to McrBC titration and assayed by the radioactive PCR with primers indicated in (A). The autoradiography of the PCR analysis is shown in (C) and quantified in (D).
(E) PCR analysis of genomic DNA from D5 Th1 and D10 Th2 cells following digestion with BamHI, B; BamHI + McrBC, B+M; BamHI + SmaI, B+S; or BamHI + XmaI, B+X, as indicated. The primers used amplified the promoter region (panel a) or the region spanning exon 1, intron 1, and exon 2 (panels b and c).
Immunity  , DOI: ( /S (02)00314-X)

6. Figure 5
Demethylation of Different Regions of the IL-4 Gene in Th2 Cells
(A) Diagram indicating the locations of PCR primers (arrows) spanning the transcription start site (TSS)/exon 1, intron 1/exon 2, and the 2.5 kb region. DH site VA is indicated as a dark gray oval. For other details, see Figure 4A.
(B) Th2 differentiation is accompanied by increasing demethylation of the 5′ region of the IL-4 gene. Naive CD4 T cells were cultured under no cytokine, Th1, or Th2 conditions as indicated. Genomic DNA was digested with McrBC and amplified with the primers shown in (A). The numbers below the lanes give intensities for the bands with the values for naive CD4 cells (top panel) and Th1 1-wk (bottom panel) set to 1. TSS/exon 1 and intron 1 PCR products were normalized to those of the site VA region. D5 and D10 DNAs were included in all experiments as controls for restriction digest and PCR product specificity (data not shown for bottom panel).
(C) Genomic DNA was purified from Th2 cultures and digested with BamHI or BamHI + McrBC. The digested DNA was subjected to PCR analysis using primers flanking three major regions of demethylation shown in part (A). The same exposure times are shown for both the right and left panels.
(D) Graphical representation of (C). The 2.5 kb region is demethylated with a distinct lag relative to the TSS/exon 1 and the intron 1/exon 2 regions.
Immunity  , DOI: ( /S (02)00314-X)

7. Figure 6
Intronic Demethylation Is Associated with High-Level IL-4 Gene Expression
(A) Naive CD4 cells from D TCR tg × RAG2−/− mice and D10 Th2 clones were stimulated with immobilized αCD3/αCD28 for 1 hr or 3 hr. Radioactive RT-PCR was performed using primers against IL-4 and HPRT mRNA.
(B) Th2 cells cultured for 18 days (restimulated once with α-CD3 at day 8) were stimulated and prepared for intracellular IL-4 cytokine staining and sorted based on low and high IL-4 expression. From top to bottom, the four flow cytometry diagrams represent unstimulated control, total stimulated population, low IL-4+ sorted cells, and high IL-4+ sorted cells.
(C) Twenty nanograms of SmaI-digested DNAs from HI and LO sorted cells were subjected to methylation analysis by PCR using intron 1 or site VA primers.
Immunity  , DOI: ( /S (02)00314-X)

8. Figure 7
Summary of the Chromatin Changes in the IL-4 Locus during Commitment of Naive T Cells to the Th2 Lineage
Two DH sites are present in naive CD4 T cells, HSS 3 and site IV. The promoter and the CNS-2 region are demethylated. Upon TCR stimulation in the presence of IL-4, Th2-specific DH sites appear throughout the IL-4/IL-13 locus, demethylation extends outward from the promoter to the transcription start site/intron 1 region, and the intergenic DH sites become demethylated. Prolonged exposure to Th2 conditions further extends demethylation in the 3′ direction into the IL-4 gene; this process correlates with efficient IL-4 transcription and serves as a marker for terminally differentiated Th2 cells. The CNS-2 region remains demethylated under Th2 conditions but slowly undergoes methylation under Th1 conditions.
Immunity  , DOI: ( /S (02)00314-X)