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Primary 1,25-dihydroxyvitamin D3 response of the interleukin 8 gene cluster in human monocyte- and macrophage-like cells.

Ryynänen J, Carlberg C - PLoS ONE (2013)

Bottom Line: Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site.In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR.Our observation provides further evidence for the immune-related functions of vitamin D.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

ABSTRACT
Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

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Detailed genomic view of VDR association and 1,25(OH)2D3-dependent chromatin opening.A. The IGV browser was used to display the genomic region around the CXCL8 gene. The peak tracks show VDR ChIP-seq data (red [32]) and FAIRE-seq data (grey for the ethanol-treated control, turquoise for the samples treated with 1,25(OH)2D3 for indicated times [51]), both from THP-1 cells. The gene structures are shown in blue. The sequence of a DR3-type VDR binding site below the summit of the VDR ChIP-seq peaks is indicated. B. ChIP-qPCR was performed with chromatin samples obtained from THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding sites close to the CXCL8 gene and a negative control region of chromosome 6. Cells were stimulated for 0, 1 and 2 h with 10 nM 1,25(OH)2D3 and chromatin was extracted. Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of 1,25(OH)2D3-induced VDR association in reference to untreated cells (** p < 0.01).
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pone-0078170-g003: Detailed genomic view of VDR association and 1,25(OH)2D3-dependent chromatin opening.A. The IGV browser was used to display the genomic region around the CXCL8 gene. The peak tracks show VDR ChIP-seq data (red [32]) and FAIRE-seq data (grey for the ethanol-treated control, turquoise for the samples treated with 1,25(OH)2D3 for indicated times [51]), both from THP-1 cells. The gene structures are shown in blue. The sequence of a DR3-type VDR binding site below the summit of the VDR ChIP-seq peaks is indicated. B. ChIP-qPCR was performed with chromatin samples obtained from THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding sites close to the CXCL8 gene and a negative control region of chromosome 6. Cells were stimulated for 0, 1 and 2 h with 10 nM 1,25(OH)2D3 and chromatin was extracted. Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of 1,25(OH)2D3-induced VDR association in reference to untreated cells (** p < 0.01).

Mentions: The FAIRE-seq pattern of the genomic region around the genes CXCL8, CXCL6 and CXCL1 suggests that a treatment with 1,25(OH)2D3 has no global effect on the number or intensity of sites of open chromatin in THP-1 cells (Figure 1A and data not shown). However, we observed at the VDR binding site close to the CXCL8 gene a significant, 1,25(OH)2D3-dependent opening of chromatin in a FAIRE-seq time course experiment with measurements every 20 min over a time period of 120 min (Figure 3A). In order to confirm VDR binding to this site, we performed ChIP-qPCR with chromatin samples from THP-1 cells that had been treated for 0, 1 and 2 h with 1,25(OH)2D3 (Figure 3B). In comparison to a negative control region from chromosome 6, we observed already in the absence of ligand VDR binding to the site, which significantly increased by the addition of 1,25(OH)2D3. From previous studies [32,50,51] we know that VDR binding sites at regions of 1,25(OH)2D3-dependent chromatin opening have genome-wide the highest rate of DR3-type response elements (66%) below VDR ChIP-seq summits. Consistent with this, the VDR binding site close to the CXCL8 gene also contained a sequence with a high similarity score to a DR3-type response element (Figure 3A).


Primary 1,25-dihydroxyvitamin D3 response of the interleukin 8 gene cluster in human monocyte- and macrophage-like cells.

Ryynänen J, Carlberg C - PLoS ONE (2013)

Detailed genomic view of VDR association and 1,25(OH)2D3-dependent chromatin opening.A. The IGV browser was used to display the genomic region around the CXCL8 gene. The peak tracks show VDR ChIP-seq data (red [32]) and FAIRE-seq data (grey for the ethanol-treated control, turquoise for the samples treated with 1,25(OH)2D3 for indicated times [51]), both from THP-1 cells. The gene structures are shown in blue. The sequence of a DR3-type VDR binding site below the summit of the VDR ChIP-seq peaks is indicated. B. ChIP-qPCR was performed with chromatin samples obtained from THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding sites close to the CXCL8 gene and a negative control region of chromosome 6. Cells were stimulated for 0, 1 and 2 h with 10 nM 1,25(OH)2D3 and chromatin was extracted. Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of 1,25(OH)2D3-induced VDR association in reference to untreated cells (** p < 0.01).
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Related In: Results  -  Collection

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pone-0078170-g003: Detailed genomic view of VDR association and 1,25(OH)2D3-dependent chromatin opening.A. The IGV browser was used to display the genomic region around the CXCL8 gene. The peak tracks show VDR ChIP-seq data (red [32]) and FAIRE-seq data (grey for the ethanol-treated control, turquoise for the samples treated with 1,25(OH)2D3 for indicated times [51]), both from THP-1 cells. The gene structures are shown in blue. The sequence of a DR3-type VDR binding site below the summit of the VDR ChIP-seq peaks is indicated. B. ChIP-qPCR was performed with chromatin samples obtained from THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding sites close to the CXCL8 gene and a negative control region of chromosome 6. Cells were stimulated for 0, 1 and 2 h with 10 nM 1,25(OH)2D3 and chromatin was extracted. Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of 1,25(OH)2D3-induced VDR association in reference to untreated cells (** p < 0.01).
Mentions: The FAIRE-seq pattern of the genomic region around the genes CXCL8, CXCL6 and CXCL1 suggests that a treatment with 1,25(OH)2D3 has no global effect on the number or intensity of sites of open chromatin in THP-1 cells (Figure 1A and data not shown). However, we observed at the VDR binding site close to the CXCL8 gene a significant, 1,25(OH)2D3-dependent opening of chromatin in a FAIRE-seq time course experiment with measurements every 20 min over a time period of 120 min (Figure 3A). In order to confirm VDR binding to this site, we performed ChIP-qPCR with chromatin samples from THP-1 cells that had been treated for 0, 1 and 2 h with 1,25(OH)2D3 (Figure 3B). In comparison to a negative control region from chromosome 6, we observed already in the absence of ligand VDR binding to the site, which significantly increased by the addition of 1,25(OH)2D3. From previous studies [32,50,51] we know that VDR binding sites at regions of 1,25(OH)2D3-dependent chromatin opening have genome-wide the highest rate of DR3-type response elements (66%) below VDR ChIP-seq summits. Consistent with this, the VDR binding site close to the CXCL8 gene also contained a sequence with a high similarity score to a DR3-type response element (Figure 3A).

Bottom Line: Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site.In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR.Our observation provides further evidence for the immune-related functions of vitamin D.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

ABSTRACT
Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

Show MeSH
Related in: MedlinePlus