Figure 5: TH2-LCR lncRNA regulates IL-4, IL-5 and IL-13 expression.
From: Expression and functions of long noncoding RNAs during human T helper cell differentiation
(a) Genomic organization of IL5, RAD50, IL13 and IL4 and the TH2-LCR lncRNA cluster (UCSC genome browser). (b) Shared and unique RNA sequences in alternatively spliced TH2-LCR lncRNAs. Black: non-shared sequences, green: shared sequences among all TH2-LCR lncRNAs, red: identify shared by three lncRNAs, orange; long and short sequence identity shared by all or two transcripts, blue: sequence identity shared by two transcripts. (c) TH2-LCR lncRNA transcripts are TH2 lineage-specific. PCR primers were designed to detect each TH2-LCR lncRNA alternatively spliced transcript by RT–PCR. Results are mean expression±s.d. after normalization to GAPDH (n=4). *P<0.05 in TH2 versus TH1 and TH17 cultures. (d) Depletion of TH2-LCR lncRNA abrogates IL-4, IL-13 and IL-5 expression by TH2 cells. TH2 cultures were transfected with a negative control siRNA (−) or siRNA (+) targeting the TH2-LCR lncRNA shared sequence (green, b). Intracellular cytokine staining was performed. Results are expressed as mean % CD4+ T cells expressing each cytokine±s.d. (n=4). *P<0.05 comparing transfections with control siRNA to TH2-LCR lncRNA-specific siRNA. (e) Effect of siRNA-mediated knockdown of individual TH2-LCR lncRNA transcripts on IL-4, IL-13 and IL-5 expression by TH2 cells. siRNAs were designed to target individual TH2-LCR lncRNAs, BK1, BK2, BK3, BK4, two TH2-LCR lncRNAs (blue) or three TH2-LCR lncRNAs (red; see b). IL-4, IL-13 and IL-5 expression was determined by intracellular cytokine staining. Results are expressed as % inhibition relative to transfection with scrambled siRNA control±s.d. (n=3). (f,g) Effects of TH2-LCR lncRNA knockdown on formation of H3K4-Me3 marks (f) and WDR5 localization (g) at IL4, IL13 and IL5 loci. Non-transfected TH1 cultures and TH2 cultures, transfected with a scrambled siRNA (TH2-sc-siRNA) or TH2-LCR lncRNA-specific siRNA (TH2-sp-siRNA) on day 3, were harvested on day 5. Levels of H3K4-Me3 or WDR5 were determined by ChIP and are expressed as a fraction of input±s.d. (n=3). *P<0.05 comparing either TH1 to TH2-sc-RNA cultures or **TH2-sc-siRNA to TH2-sp-siRNA cultures. (h) Effect of TH2-LCR lncRNA knockdown on RAD50 expression. As in d, except RNA was isolated and RAD50 transcript levels±s.d. determined after normalization to GAPDH (n=3). P values were determined using Student’s t-test.
