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Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms.

Turner JD, Pelascini LP, Macedo JA, Muller CP - Nucleic Acids Res. (2008)

Bottom Line: The majority of positions were methylated at levels >25% in at least one donor affecting each promoter and TSS.We also examined the evolutionarily conserved transcription factor binding sites (TFBS) using an improved in silico phylogenetic footprinting technique.The heterogeneity observed may reflect epigenetic mechanisms of GR fine tuning, programmed by early life environment and events.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Laboratoire National de Santé, Luxembourg, Germany.

ABSTRACT
The transcription start sites (TSS) and promoters of many genes are located in upstream CpG islands. Methylation within such islands is known for both imprinted and oncogenes, although poorly studied for other genes, especially those with complex CpG islands containing multiple first exons and promoters. The glucocorticoid receptor (GR) CpG island contains seven alternative first exons and their promoters. Here we show for the five GR promoters activated in PBMCs that methylation patterns are highly variable between individuals. The majority of positions were methylated at levels >25% in at least one donor affecting each promoter and TSS. We also examined the evolutionarily conserved transcription factor binding sites (TFBS) using an improved in silico phylogenetic footprinting technique. The majority of these contain methylatable CpG sites, suggesting that methylation may orchestrates alternative first exon usage, silencing and controlling tissue-specific expression. The heterogeneity observed may reflect epigenetic mechanisms of GR fine tuning, programmed by early life environment and events. With 78% of evolutionarily conserved alternative first exons falling into such complex CpG islands, their internal structure and epigenetic modifications are bound to be biologically important, and may be a common transcriptional control mechanism used throughout many phyla.

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Related in: MedlinePlus

In silico phylogenetic footprinting and bisulphite sequencing of promoters 1J and 1E. (A) In silico phylogenetic footprinting covers the end of exon 1D through promoter 1J, exon 1J, to exon 1E. (B) Percentage methylation was measured by direct electropherogram reading after bisulphite sequencing of 26 donors, covering CpG 16–27. Methylation levels are expressed by colour, levels from the scale at the panel top. (C) CpG identifiers and the unsequenced region of promoter 1J (grey background). Exon 1J is underlined. (D) TRANSFAC prediction for promoter 1J and E. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.
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Figure 3: In silico phylogenetic footprinting and bisulphite sequencing of promoters 1J and 1E. (A) In silico phylogenetic footprinting covers the end of exon 1D through promoter 1J, exon 1J, to exon 1E. (B) Percentage methylation was measured by direct electropherogram reading after bisulphite sequencing of 26 donors, covering CpG 16–27. Methylation levels are expressed by colour, levels from the scale at the panel top. (C) CpG identifiers and the unsequenced region of promoter 1J (grey background). Exon 1J is underlined. (D) TRANSFAC prediction for promoter 1J and E. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.

Mentions: Bisulphite sequences of 269 bp covering the complete promoter 1E, exon 1J and part of promoter 1J were obtained (Figure 1). Of the 28 potential CpG methylation sites, 12 were successfully sequenced: four in promoter 1J, four in exon 1J, and four in the promoter region immediately upstream of exon 1E (Figure 3). Each of these sites (except CpG27) was methylated in one to three of the 26 donors. Interestingly, the in silico analysis identified nine TFBS that are evolutionarily highly conserved and present in at least four of the five species. However, only two of these nine sites contain CpG di-nucleotides: Pax3 (-4067) in promoter 1J (CpG16), and Sp3 (-4012) in promoter 1E (CpG19). Bisulphite sequencing of these nucleotides showed that whilst methylation was possible it was nevertheless rare (one and three donors, respectively at levels below 50%).Figure 3.


Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms.

Turner JD, Pelascini LP, Macedo JA, Muller CP - Nucleic Acids Res. (2008)

In silico phylogenetic footprinting and bisulphite sequencing of promoters 1J and 1E. (A) In silico phylogenetic footprinting covers the end of exon 1D through promoter 1J, exon 1J, to exon 1E. (B) Percentage methylation was measured by direct electropherogram reading after bisulphite sequencing of 26 donors, covering CpG 16–27. Methylation levels are expressed by colour, levels from the scale at the panel top. (C) CpG identifiers and the unsequenced region of promoter 1J (grey background). Exon 1J is underlined. (D) TRANSFAC prediction for promoter 1J and E. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602793&req=5

Figure 3: In silico phylogenetic footprinting and bisulphite sequencing of promoters 1J and 1E. (A) In silico phylogenetic footprinting covers the end of exon 1D through promoter 1J, exon 1J, to exon 1E. (B) Percentage methylation was measured by direct electropherogram reading after bisulphite sequencing of 26 donors, covering CpG 16–27. Methylation levels are expressed by colour, levels from the scale at the panel top. (C) CpG identifiers and the unsequenced region of promoter 1J (grey background). Exon 1J is underlined. (D) TRANSFAC prediction for promoter 1J and E. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.
Mentions: Bisulphite sequences of 269 bp covering the complete promoter 1E, exon 1J and part of promoter 1J were obtained (Figure 1). Of the 28 potential CpG methylation sites, 12 were successfully sequenced: four in promoter 1J, four in exon 1J, and four in the promoter region immediately upstream of exon 1E (Figure 3). Each of these sites (except CpG27) was methylated in one to three of the 26 donors. Interestingly, the in silico analysis identified nine TFBS that are evolutionarily highly conserved and present in at least four of the five species. However, only two of these nine sites contain CpG di-nucleotides: Pax3 (-4067) in promoter 1J (CpG16), and Sp3 (-4012) in promoter 1E (CpG19). Bisulphite sequencing of these nucleotides showed that whilst methylation was possible it was nevertheless rare (one and three donors, respectively at levels below 50%).Figure 3.

Bottom Line: The majority of positions were methylated at levels >25% in at least one donor affecting each promoter and TSS.We also examined the evolutionarily conserved transcription factor binding sites (TFBS) using an improved in silico phylogenetic footprinting technique.The heterogeneity observed may reflect epigenetic mechanisms of GR fine tuning, programmed by early life environment and events.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Laboratoire National de Santé, Luxembourg, Germany.

ABSTRACT
The transcription start sites (TSS) and promoters of many genes are located in upstream CpG islands. Methylation within such islands is known for both imprinted and oncogenes, although poorly studied for other genes, especially those with complex CpG islands containing multiple first exons and promoters. The glucocorticoid receptor (GR) CpG island contains seven alternative first exons and their promoters. Here we show for the five GR promoters activated in PBMCs that methylation patterns are highly variable between individuals. The majority of positions were methylated at levels >25% in at least one donor affecting each promoter and TSS. We also examined the evolutionarily conserved transcription factor binding sites (TFBS) using an improved in silico phylogenetic footprinting technique. The majority of these contain methylatable CpG sites, suggesting that methylation may orchestrates alternative first exon usage, silencing and controlling tissue-specific expression. The heterogeneity observed may reflect epigenetic mechanisms of GR fine tuning, programmed by early life environment and events. With 78% of evolutionarily conserved alternative first exons falling into such complex CpG islands, their internal structure and epigenetic modifications are bound to be biologically important, and may be a common transcriptional control mechanism used throughout many phyla.

Show MeSH
Related in: MedlinePlus