Immune Netw.  2016 Jun;16(3):176-182. 10.4110/in.2016.16.3.176.

Roles of RUNX1 and PU.1 in CCR3 Transcription

• Kong SK
• Kim BS
• Hwang SM
• Lee HH
• Chung IY

• KMID: 2299791
• DOI: http://doi.org/10.4110/in.2016.16.3.176

Abstract

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.

Keyword

CCR3; RUNX1; PU.1; Transcription factor; Cis-acting element

MeSH Terms

Eosinophils
Mutagenesis
RNA, Small Interfering
T-Lymphocytes
Th2 Cells
Transcription Factors
Transcriptional Activation
Transfection
RNA, Small Interfering
Transcription Factors

Figure

• 

  Figure 1 Expression of CCR3, RUNX1, and PU.1 in Jurkat and EoL-1 cells. (A) Expression of CCR3 mRNA and protein was analyzed using RT-PCR and FACS, respectively, and chemotactic responses to eotaxin (1~200 ng/ml) was analyzed. The results shown are representative of three to five independent experiments. Statistical significance was obtained using SPSS, compared with medium. *p<0.05 and **p<0.01. (B) Expression of RUNX1 and PU.1 proteins was analyzed using Western blotting. GAPDH mRNA and protein were used as loading controls. The results shown are representative of three independent experiments.

• 

  Figure 2 Effects of RUNX1 and PU.1 siRNAs on CCR3 reporter activity. Cells were co-transfected with 50 nM siRNAs for RUNX1, PU.1, GATA-1 or scramble along with the CCR3 reporter. After 36 h, luciferase activity was measured and normalized to Renilla luciferase activity. Data represent the mean±SEM of three independent experiments. Statistical significance was obtained using ANOVA, compared with scrambled siRNA. *p<0.01.

• 

  Figure 3 Effects of point mutants of RUNX1 and PU.1 binding motifs on CCR3 reporter activity. (A) Nucleotide sequence of the regulatory region construct 1 containing exon 1 and proximal intron 1 of the CCR3 gene. The sites for RUNX1 and PU.1 are underlined, as is the functional GATA site. (B) Point mutants were constructed and transfected into EoL-1 and Jurkat cells. Reporter activity was measured 48 h post-transfection. Transfection efficiency was normalized to cotransfected Renilla luciferase activity. Open and filled symbols indicate wild-type and mutant sites, respectively. Data represent the mean±SEM of three independent experiments performed in triplicate. *p<0.01.

• 

  Figure 4 Binding of RUNX1 and PU.1 to their putative sites on the CCR3 regulatory region. Nuclear extracts of EoL-1 and Jurkat cells were incubated with probes harboring RUNX1 and PU.1 binding elements in the presence or absence of their respective Abs. S and C indicate specific Ab and control Ab, respectively. *, **, and *** indicate specific binding, a supershift, and non-specific binding, respectively.

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