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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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Genome view of the CXCL gene cluster.A. The IGV browser was used to show the peak tracks of FAIRE-seq data from THP-1 cells [51] (stimulated for 20 min with ethanol, turquoise) and VDR ChIP-seq data from THP-1 cells [32] (unstimulated (-) and treated for 40 min with 1,25(OH)2D3 (+), red). The gene structures are shown in blue and the 9 genes of the CXCL gene cluster are underlayed in grey. The THP-1 data were compared with CTCF ChIP-seq data from the ENCODE cell lines K562, HUVEC and NHEK [42] (blue) and CTCF ChIA-PET data [47] in track view (dark blue) and in looping view (grey horizontal lines). Six conserved CTCF sites were highlighted. B. ChIP-qPCR was performed with chromatin samples obtained from unstimulated THP-1 cells to determine CTCF (blue) and unspecific IgG (grey) binding at the six genomic regions, which were suggested by data obtained in K562 cells (see panel A). 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 CTCF association in reference to a control region from chromosome 6 (* p < 0.05; ** p < 0.01; *** p  < 0.001).
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pone-0078170-g001: Genome view of the CXCL gene cluster.A. The IGV browser was used to show the peak tracks of FAIRE-seq data from THP-1 cells [51] (stimulated for 20 min with ethanol, turquoise) and VDR ChIP-seq data from THP-1 cells [32] (unstimulated (-) and treated for 40 min with 1,25(OH)2D3 (+), red). The gene structures are shown in blue and the 9 genes of the CXCL gene cluster are underlayed in grey. The THP-1 data were compared with CTCF ChIP-seq data from the ENCODE cell lines K562, HUVEC and NHEK [42] (blue) and CTCF ChIA-PET data [47] in track view (dark blue) and in looping view (grey horizontal lines). Six conserved CTCF sites were highlighted. B. ChIP-qPCR was performed with chromatin samples obtained from unstimulated THP-1 cells to determine CTCF (blue) and unspecific IgG (grey) binding at the six genomic regions, which were suggested by data obtained in K562 cells (see panel A). 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 CTCF association in reference to a control region from chromosome 6 (* p < 0.05; ** p < 0.01; *** p  < 0.001).

Mentions: Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) is a method that allows the identification of chromatin sites devoid of nucleosomes, roughly translating to the genome-wide localization of chromatin regions that are accessible to transcription factors, such as VDR, at a given time and condition [48,49]. In this study, we used a FAIRE-seq dataset obtained from THP-1 human monocytic leukemia cells [50,51] and aligned the resulting peaks with the VDR ChIP-seq dataset from the same cell line [32]. Interestingly, a chromatin region spanning over 180 kb (from 45 kb upstream of the CXCL8 gene to 9 kb downstream of the CXCL1 gene, underlined in the top lane of Figure 1A) displayed a higher rate of open chromatin, since it showed stronger FAIRE signals than its up- and downstream flanking regions. VDR ChIP-seq analysis in THP-1 cells [32] of the same genomic region around the CYCL cluster highlighted a prominent, 1,25(OH)2D3-inducible VDR binding site 22 kb downstream of the transcription start site (TSS) of the CXCL8 gene (Figure 1A).


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)

Genome view of the CXCL gene cluster.A. The IGV browser was used to show the peak tracks of FAIRE-seq data from THP-1 cells [51] (stimulated for 20 min with ethanol, turquoise) and VDR ChIP-seq data from THP-1 cells [32] (unstimulated (-) and treated for 40 min with 1,25(OH)2D3 (+), red). The gene structures are shown in blue and the 9 genes of the CXCL gene cluster are underlayed in grey. The THP-1 data were compared with CTCF ChIP-seq data from the ENCODE cell lines K562, HUVEC and NHEK [42] (blue) and CTCF ChIA-PET data [47] in track view (dark blue) and in looping view (grey horizontal lines). Six conserved CTCF sites were highlighted. B. ChIP-qPCR was performed with chromatin samples obtained from unstimulated THP-1 cells to determine CTCF (blue) and unspecific IgG (grey) binding at the six genomic regions, which were suggested by data obtained in K562 cells (see panel A). 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 CTCF association in reference to a control region from chromosome 6 (* p < 0.05; ** p < 0.01; *** p  < 0.001).
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pone-0078170-g001: Genome view of the CXCL gene cluster.A. The IGV browser was used to show the peak tracks of FAIRE-seq data from THP-1 cells [51] (stimulated for 20 min with ethanol, turquoise) and VDR ChIP-seq data from THP-1 cells [32] (unstimulated (-) and treated for 40 min with 1,25(OH)2D3 (+), red). The gene structures are shown in blue and the 9 genes of the CXCL gene cluster are underlayed in grey. The THP-1 data were compared with CTCF ChIP-seq data from the ENCODE cell lines K562, HUVEC and NHEK [42] (blue) and CTCF ChIA-PET data [47] in track view (dark blue) and in looping view (grey horizontal lines). Six conserved CTCF sites were highlighted. B. ChIP-qPCR was performed with chromatin samples obtained from unstimulated THP-1 cells to determine CTCF (blue) and unspecific IgG (grey) binding at the six genomic regions, which were suggested by data obtained in K562 cells (see panel A). 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 CTCF association in reference to a control region from chromosome 6 (* p < 0.05; ** p < 0.01; *** p  < 0.001).
Mentions: Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) is a method that allows the identification of chromatin sites devoid of nucleosomes, roughly translating to the genome-wide localization of chromatin regions that are accessible to transcription factors, such as VDR, at a given time and condition [48,49]. In this study, we used a FAIRE-seq dataset obtained from THP-1 human monocytic leukemia cells [50,51] and aligned the resulting peaks with the VDR ChIP-seq dataset from the same cell line [32]. Interestingly, a chromatin region spanning over 180 kb (from 45 kb upstream of the CXCL8 gene to 9 kb downstream of the CXCL1 gene, underlined in the top lane of Figure 1A) displayed a higher rate of open chromatin, since it showed stronger FAIRE signals than its up- and downstream flanking regions. VDR ChIP-seq analysis in THP-1 cells [32] of the same genomic region around the CYCL cluster highlighted a prominent, 1,25(OH)2D3-inducible VDR binding site 22 kb downstream of the transcription start site (TSS) of the CXCL8 gene (Figure 1A).

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