American Society of Hematology

Fig. 2.

$Fig. 2. Increased accessibility of the CCR3 gene in Th2 cells. / (A) AZA and TSA induce CCR3 expression in activated T cells. Purified naive CD4+ T cells were first stimulated with α-CD3 + α-CD28 in the presence of IL-2 and IL-4. After 10 days, the cells were restimulated under the same conditions in the presence of either 5 μM AZA or 16.5 nM TSA or both drugs (AZA + TSA). After 3 days the cells were washed and expanded for 5 days with IL-2. Semiquantitative PCR experiments were performed with cDNA dilutions (1/5, 1/25, and 1/125). The result of CCR3 amplifications was visualized by Southern blot. β-actin amplification was analyzed by ethidium bromide staining of the agarose gel. The increased receptor expression was also detected by cell surface staining (not shown). (B) Two regions of the human CCR3 gene are associated with acetylated H3 histones in Th2 cells. Primers used are described in “Study design.” The first set (1) covers 251 bp of the promoter region from −239 to +13, the second set (2) covers 339 bp of the upstream intronic region from +9284 to +9623, and the third set (3) covers 189 bp of the downstream intronic region from +20 540 to +20 729. Soluble chromatin was immunoprecipitated with an anti–acetyl H3 antibody from CCR3+ and CCR3− T cells. DNA was purified from the different fractions (input, unbound, bound), and equal amounts were amplified by PCR under stringent and nonsaturating conditions (25 cycles). PCR products were visualized by Southern blot by using specific probes generated by PCR. Similar results were obtained in 3 independent experiments. (C) Increased accessibility to DNaseI of CCR3 gene in Th2 cells. Nuclei were purified from 1 to 2.107 cells and digested with increasing concentrations of DNaseI. Genomic DNA was purified, and each fraction was digested by HindIII. The results were visualized by Southern blot. Left panel: DNAseI HS analysis of the CCR3 promoter region in a Th2 line and Jurkat cells. Membranes were hybridized using a 3′ probe (2) of 339 bp (from +9284 to +9623). The 6.9-kbHindIII fragment analyzed and the 1.9-kb HS site are indicated with arrows. Right panel: DNAseI HS analysis of the 3′ intronic region of CCR3. Nuclei were purified from CCR3+(left) and CCR3− (right) cells, and membranes were hybridized using a probe (3) of 796 bp (from +20 540 to +21 336). The 5.678-kb HindIII fragment analyzed and the 2.7-kb HS site are indicated.$

Increased accessibility of the CCR3 gene in Th2 cells.

(A) AZA and TSA induce CCR3 expression in activated T cells. Purified naive CD4⁺ T cells were first stimulated with α-CD3 + α-CD28 in the presence of IL-2 and IL-4. After 10 days, the cells were restimulated under the same conditions in the presence of either 5 μM AZA or 16.5 nM TSA or both drugs (AZA + TSA). After 3 days the cells were washed and expanded for 5 days with IL-2. Semiquantitative PCR experiments were performed with cDNA dilutions (1/5, 1/25, and 1/125). The result of CCR3 amplifications was visualized by Southern blot. β-actin amplification was analyzed by ethidium bromide staining of the agarose gel. The increased receptor expression was also detected by cell surface staining (not shown). (B) Two regions of the human CCR3 gene are associated with acetylated H3 histones in Th2 cells. Primers used are described in “Study design.” The first set (1) covers 251 bp of the promoter region from −239 to +13, the second set (2) covers 339 bp of the upstream intronic region from +9284 to +9623, and the third set (3) covers 189 bp of the downstream intronic region from +20 540 to +20 729. Soluble chromatin was immunoprecipitated with an anti–acetyl H3 antibody from CCR3⁺ and CCR3⁻ T cells. DNA was purified from the different fractions (input, unbound, bound), and equal amounts were amplified by PCR under stringent and nonsaturating conditions (25 cycles). PCR products were visualized by Southern blot by using specific probes generated by PCR. Similar results were obtained in 3 independent experiments. (C) Increased accessibility to DNaseI of CCR3 gene in Th2 cells. Nuclei were purified from 1 to 2.10⁷ cells and digested with increasing concentrations of DNaseI. Genomic DNA was purified, and each fraction was digested by HindIII. The results were visualized by Southern blot. Left panel: DNAseI HS analysis of the CCR3 promoter region in a Th2 line and Jurkat cells. Membranes were hybridized using a 3′ probe (2) of 339 bp (from +9284 to +9623). The 6.9-kbHindIII fragment analyzed and the 1.9-kb HS site are indicated with arrows. Right panel: DNAseI HS analysis of the 3′ intronic region of CCR3. Nuclei were purified from CCR3⁺(left) and CCR3⁻ (right) cells, and membranes were hybridized using a probe (3) of 796 bp (from +20 540 to +21 336). The 5.678-kb HindIII fragment analyzed and the 2.7-kb HS site are indicated.

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