Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity

<div><p>Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in <i>in vivo</i> generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.</p></div

The microarray analysis revealed the potential GATA3-regulated genes in Th2 cells.

<p>(<b>A</b>), Effector Th2 cells (1^st) and fully developed Th2 cells (4^th) were treated with GATA3 siRNA and their gene expression profiles were studied and compared to cells treated with control siRNA using a microarray analysis. In the upper panel, the lower row indicates the number of genes that showed more than two-fold GATA3/Control siRNA ratios in the Th2-1^st cells. The middle row indicates 0.5 to two-fold GATA3/Control siRNA ratios and the upper row indicates less than 0.5-fold ratios. The right column indicates the number of genes that showed more than two-fold GATA3/Control siRNA ratios in Th2-4^th cells. The center indicates a 0.5 to two-fold GATA3/Control siRNA ratios and the left column indicates ratios of less than 0.5-fold. A total of 55 genes were identified as being positively regulated by GATA3 in both Th2 and Th2-4^th cells in the microarray analysis. Another 58 genes were determined to be negatively regulated by GATA3 in both Th2 and Th2-4^th cells. The right heat map shows the classification of the 55 common positively regulated genes and the 58 common negatively regulated genes. The black and gray bars indicate the genes in which GATA3 binding was detected in the ChIP-seq analysis. Among these 55 positively regulated genes, 10 GATA3 target genes were classified as Th2-high genes and seven GATA3 target genes were classified as Th1-high genes. (<b>B</b>), The effects of GATA3 knockdown on the genes negatively regulated by GATA3 in Th2-1^st and Th2-4^th cells were evaluated with qRT-PCR. The relative expression (GATA3/Control siRNA) levels are shown. The lower dashed line indicates 10^-1/2 and the upper dashed line indicates 10^1/2. (<b>C</b>), The effects of GATA3 knockdown on the genes positively regulated by GATA3 in Th2-1^st and Th2-4^th cells were evaluated with qRT-PCR. The relative expression (GATA3/Control siRNA) levels are shown. A number sign (#) with the gene name indicates data that are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066468#pone-0066468-g001" target="_blank">Figure 1</a>.</p

The results of the microarray analysis revealed the GATA3-regulated cytokine and cytokine receptor genes in Th2 cells.

<p>(<b>A</b>), The names of the selected 24 cytokine genes. The decreased expression of four genes (Cxcl3, Il-5, Il-6 and Tnfsf10) was observed in both Th2-1^st and Th2-4^th cells. GATA3 siRNA reduced the expression of two genes (Il-13 and Il-24) in Th2-1^st cells and six genes (Areg, Bmp2, Bmp7, Ccl20, Ccl28 and Lif) in Th2-1^st cells. The increased expression of two genes (Lta and Il-33) was observed in both Th2 and Th2-4^th cells. GATA3 siRNA increased the expression of eight genes (Ccl9, Cd70, Clcf1, Cxcl10, Ltb, Tnf, Tnfsf9 and Tnfsf11) in Th2-1^st cells and two genes (Ccl2 and Ccl12) in Th2-4^th cells. (<b>B</b>), The names of 11 selected cytokine receptor genes. GATA3 siRNA reduced the expression of five genes (Ccr1, Ccr8, Ccr9, Cxcr6 and Il-13ra) in Th2-1^st cells, and two genes (Cxcr4 and Il-10ra) in Th2-4^th cells. The increased expression of one gene (Il-1rl1) was observed in both Th2-1^st and Th2-4^th cells. GATA3 siRNA increased the expression of two genes (Il-1rap and Il-18rap) in Th2-1^st cells and one gene (Ccr5) in Th2-4^th cells.</p

The effects of GATA3 knockdown on the expression of cytokine and cytokine receptor genes.

<p>The effects of GATA3 knockdown on cytokine (A) and cytokine receptor (B) gene expression in Th2-1^st and Th2-4^th cells were evaluated with qRT-PCR. The relative expression (GATA3/Control siRNA) levels of the cytokine and cytokine receptor genes are shown. A number sign (#) with the gene name indicates data that are also shown in previous Figures. The filled triangles indicate the genes that showed GATA3 dependency changes (more than two-fold) in Th2-4^th cells compared with that observed in Th2-1^st cells. The lower dashed line indicates 10^-1/2 and the upper dashed line indicates 10^1/2.</p

The effects of GATA3 knockdown on the gene expression of Th2-1^st, Th2-4^th and memory Th2 cells.

<p>The effects of GATA3 knockdown on effector Th2-1^st (A), Th2-4^th (B) and memory Th2 cells (C) were determined with qRT-PCR. The relative expression (GATA3/Control siRNA) levels are shown. The expression data were rank-ordered from genes with the highest to the lowest relative expression ratios (GATA3/Control siRNA) in Th2-1^st cells. The filled squares indicate genes that showed decreases in GATA3 dependency in Th2-memory cells compared with that observed in Th2-1^st cells. The open squares indicate genes that showed increases in GATA3 dependency in Th2-memory cells compared with that observed in Th2-1^st cells. The lower dashed line indicates 10^-1/2 and the upper dashed line indicates 10^1/2.</p

(A) mRNA expression of and p53-related proapoptotic genes in effector Th2 cells was determined by quantitative RT-PCR

The relative intensity (/HPRT; mean of three samples) is shown with standard deviations. Three independent experiments were performed with similar results. (B) mRNA expression of , p53-related proapoptotic genes, and antiapoptotic genes in memory Th2 cells was analyzed. The relative intensity (/HPRT; mean of three samples) is shown with standard deviations. Two independent experiments were performed with similar results. (C) Effects on p16 and p19 deficiency on the memory Th2 cell generation. The effector Th2 cells from the indicated mice (Ly5.2 background) were transferred into Ly5.1 host mice. 5 wk after cell transfer, the number of Ly5.2 memory Th2 cells was determined. The mean values are shown with standard deviations ( = 5; right). The experiments were performed twice with similar results. (D) mRNA levels of proapoptotic genes in // effector Th2 cells were determined by quantitative RT-PCR. The relative intensity (/HPRT; mean of three samples) is shown with standard deviations. The experiments were performed twice with similar results.<p><b>Copyright information:</b></p><p>Taken from "Bmi1 regulates memory CD4 T cell survival via repression of the gene"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1109-1120.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373843.</p><p></p

(A) Enforced expression of Noxa-induced cell death in effector Th2 cells after cytokine depletion

Effector Th2 cells infected with a –containing retrovirus were cultured in vitro for 24 h without cytokines. hNGFR profiles (left) and annexin V staining profiles of the electronically gated hNGFR (gate #2) and hNGFR (gate #1) populations are shown. Three independent experiments were performed with similar results. (B) KJ1 effector Th2 cells infected with –containing retrovirus were transferred into BALB/c mice. 5 wk later, memory Th2 cell generation was determined by KJ1/EGFP expression. Expression of EGFP in pretransferred effector Th2 cells (top left) and a typical KJ1/GFP profile of freshly prepared memory Th2 cells (top right) are shown. In the bottom panels, the percentages of KJ1 cells and GFP Noxa-overexpressing cells and the mean fluorescence intensity of the GFP cells are shown with standard deviations ( = 4). The experiments were performed twice with similar results. (C) The effector Th2 cells from /, /, and / mice (Ly5.2) were transferred into Ly5.1 host mice, and the number of Ly5.2 memory Th2 cells was determined. A typical staining pattern of CD4/Ly5.2 (top) and the percentages of Ly5.2 cells among CD4 T cells are shown with standard deviations ( = 5; bottom). Three independent experiments were performed with similar results. (D) Deletion of the gene enhanced the generation of memory Th2 cells. In vitro–generated effector Th2 cells (Ly5.2) were transferred into Ly5.1 host mice. 5 wk after cell transfer, the number of Ly5.2 memory Th2 cells was determined. A representative CD4/Ly5.2 profile (left) and the mean values with standard deviations ( = 5; right) are shown. The experiments were performed twice with similar results.<p><b>Copyright information:</b></p><p>Taken from "Bmi1 regulates memory CD4 T cell survival via repression of the gene"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1109-1120.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373843.</p><p></p

Overview of 32 Th2-specific inducible genes, highlighting their functions and GATA3 dependency.

<p>(*) GATA3 dependency was evaluated by the recovery score (RS) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066468#pone.0066468-Horiuchi1" target="_blank">[22]</a>.</p><p>(**) GATA3 dependency was evaluated by gene expression ratio (Gata3/Control siRNA).</p><p>“Unevaluable” means that mRNA level was too low to detect by qPCR.</p><p>Unifying 24 genes identified in the present study and previously reported 26 genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066468#pone.0066468-Horiuchi1" target="_blank">[22]</a>, 32 genes were defined as Th2-specific inducible genes. Four biological readouts in these 32 genes are shown; (1) changes in gene expression by enforced expression of hGATA3 in STAT6-deficient Th2-1^st cells (3^rd column), (2) changes in gene expression by GATA3 knockdown in Th2-1^st cells (4^th column), (3) those in Th2-4^th cells (5^th column), and (4) those in memory Th2 cells (6^th column). In the 3^rd column, GATA3 dependency was determined by the recovery score (RS), which is defined as the linear equation below <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066468#pone.0066468-Horiuchi1" target="_blank">[22]</a>:</p><p>RS (%)  = 100×(C−B)/(A−B).</p><p>where <i>A</i> indicates the signal intensity of Th2, <i>B</i> indicates the signal intensity of STAT6-deficient Th2, and <i>C</i> indicates the signal intensity of STAT6-deficient Th2 with overexpression of hGATA3. In the 4^th, 5^th, and 6^th column, GATA3 dependency was determined by gene expression ratio (<i>Gata3</i>/Control siRNA). Gene ontology (GO) terms identified by DAVID analysis tool are shown in the 7^th column.</p

Decreases in GATA3 dependency correlate with increases in the gene expression.

<p>(<b>A</b>), The effects of GATA3 knockdown on effector Th2 cells (1st) and fully developed Th2 cells (2nd, 3rd and 4th) were determined with qRT-PCR. The % expression (GATA3/Control siRNA) was found to be slightly increased in Il-5, Il-13, Cxcl3 and Cxcr6 in accordance with the culture cycle number. (<b>B</b>), The gene expression levels of Th2 cells (1st) and fully developed Th2 cells (2nd, 3rd and 4th) were determined with qRT-PCR. The gene expression levels of Il-5, Il-13, Cxcl3 and Cxcr6 were increased in fully developed Th2 cells compared with those observed in effector Th2 cells.</p

The effects of GATA3 knockdown on the expression of Th2-specific genes.

<p><b>(A),</b> GATA3 target and non-target genes were classified into nine groups according to their H3K9Ac and expression levels in Th2 cells. The H3K9Ac levels were determined according to the total tag count of H3K9Ac across the gene body of each GATA3-target and GATA3-non-target RefSeq gene in Th2 versus Th1 cells. The right column indicates the number of genes that showed more than 1.5-fold Th2/Th1 ratios for the H3K9Ac level. The center indicates 1/1.5 to 1.5-fold Th2/Th1 ratios and the left column indicates genes with ratios of less than 1/1.5-fold. The transcription levels were determined using a microarray analysis that compared Th2 to Th1 cells. The lower row indicates the number of genes that showed more than two-fold Th2/Th1 ratios in expression. The middle row indicates 0.5 to two-fold Th2/Th1 ratios and the upper row indicates less than 0.5-fold ratios. From this analysis, 28 potential Th2-specific GATA3 target genes and 114 potential Th2-specific GATA3 non-target genes were identified (lower right squares in each table). The 28 potential Th2-specific GATA3 target genes and the 114 potential Th2-specific GATA3 non-target genes were validated with qRT-PCR. Next, Th2-high expression (Th2/Th1>4) and inducible (Th2/Fresh-CD4>2) genes were selected. Finally, 11 Th2-specific GATA3 non-target genes and 13 GATA3 target genes were identified. <b>(B),</b> The effects of GATA3 knockdown on Th2-1st and fully developed Th2 cells (Th2-4^th) were evaluated. The mRNA expression of the Th2-specific genes in these cells was determined with qRT-PCR. The relative expression (GATA3/Control siRNA) levels are shown. The lower dashed line indicates 10^−1/2 and the upper dashed line indicates 10^1/2.</p