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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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1,25(OH)2D3-dependent VDR association in PMA-differentiated THP-1 cells.A. The IGV browser was used to display the genomic region +/-150 kb around the VDR peak close to the CXCL8 gene. The peak tracks show VDR ChIP-seq data obtained from undifferentiated THP-1 cells (red [32]) and from PMA-differentiated THP-1 cells (green). The gene structures are shown in blue. B. ChIP-qPCR was performed with chromatin samples obtained from PMA-differentiated THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding site 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 but could not determine significant 1,25(OH)2D3-induced VDR association in reference to untreated cells.
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pone-0078170-g005: 1,25(OH)2D3-dependent VDR association in PMA-differentiated THP-1 cells.A. The IGV browser was used to display the genomic region +/-150 kb around the VDR peak close to the CXCL8 gene. The peak tracks show VDR ChIP-seq data obtained from undifferentiated THP-1 cells (red [32]) and from PMA-differentiated THP-1 cells (green). The gene structures are shown in blue. B. ChIP-qPCR was performed with chromatin samples obtained from PMA-differentiated THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding site 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 but could not determine significant 1,25(OH)2D3-induced VDR association in reference to untreated cells.

Mentions: In order to investigate, whether a differentiation of THP-1 cells into macrophage-like cells modulates the VDR binding to the CXCL gene cluster, we performed ChIP-seq for VDR in PMA-differentiated THP-1 cells. In differentiated THP-1 cells we found VDR binding at the same location than in undifferentiated cells (Figure 5A). Moreover, by ChIP-qPCR in PMA-differentiated THP-1 cells we could confirm a ligand-dependent binding of VDR to this site (Figure 5B). Furthermore, we could not detect any additional significant VDR binding site within 3 Mb distance to the CXCL cluster, when the THP-1 cells were differentiated into macrophage-like cells (Figure 5A and data not shown).


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)

1,25(OH)2D3-dependent VDR association in PMA-differentiated THP-1 cells.A. The IGV browser was used to display the genomic region +/-150 kb around the VDR peak close to the CXCL8 gene. The peak tracks show VDR ChIP-seq data obtained from undifferentiated THP-1 cells (red [32]) and from PMA-differentiated THP-1 cells (green). The gene structures are shown in blue. B. ChIP-qPCR was performed with chromatin samples obtained from PMA-differentiated THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding site 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 but could not determine significant 1,25(OH)2D3-induced VDR association in reference to untreated cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824026&req=5

pone-0078170-g005: 1,25(OH)2D3-dependent VDR association in PMA-differentiated THP-1 cells.A. The IGV browser was used to display the genomic region +/-150 kb around the VDR peak close to the CXCL8 gene. The peak tracks show VDR ChIP-seq data obtained from undifferentiated THP-1 cells (red [32]) and from PMA-differentiated THP-1 cells (green). The gene structures are shown in blue. B. ChIP-qPCR was performed with chromatin samples obtained from PMA-differentiated THP-1 cells to determine VDR association (red) and unspecific IgG binding (grey) at the VDR binding site 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 but could not determine significant 1,25(OH)2D3-induced VDR association in reference to untreated cells.
Mentions: In order to investigate, whether a differentiation of THP-1 cells into macrophage-like cells modulates the VDR binding to the CXCL gene cluster, we performed ChIP-seq for VDR in PMA-differentiated THP-1 cells. In differentiated THP-1 cells we found VDR binding at the same location than in undifferentiated cells (Figure 5A). Moreover, by ChIP-qPCR in PMA-differentiated THP-1 cells we could confirm a ligand-dependent binding of VDR to this site (Figure 5B). Furthermore, we could not detect any additional significant VDR binding site within 3 Mb distance to the CXCL cluster, when the THP-1 cells were differentiated into macrophage-like cells (Figure 5A and data not shown).

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