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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: 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.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.

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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In silico phylogenetic footprinting and bisulphite sequencing of promoter 1F. (A) In silico phylogenetic footprinting covers the end of exon 1B through to exon 1F. (B) Percentage methylation was measured by direct electropherogram reading (CpG1–30), or sequencing of 10–12 clones per donor (CpG31–43) after bisulphite sequencing of 26 donors, covering CpG 1–42. Methylation levels are expressed by colour, levels from the scale at the panel top. Blank squares indicate positions for which no data was obtainable. (C) CpG identifiers within the sequence of promoter 1F. (D) TRANSFAC prediction for promoter 1F. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.
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Figure 4: In silico phylogenetic footprinting and bisulphite sequencing of promoter 1F. (A) In silico phylogenetic footprinting covers the end of exon 1B through to exon 1F. (B) Percentage methylation was measured by direct electropherogram reading (CpG1–30), or sequencing of 10–12 clones per donor (CpG31–43) after bisulphite sequencing of 26 donors, covering CpG 1–42. Methylation levels are expressed by colour, levels from the scale at the panel top. Blank squares indicate positions for which no data was obtainable. (C) CpG identifiers within the sequence of promoter 1F. (D) TRANSFAC prediction for promoter 1F. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.

Mentions: ISPF predicted for promoter 1F several high quality potential TFBS, including the NGFI-A-binding site reported before (25,35). Since methylation of the corresponding NGFI-A-binding site has been well documented in the rat, we tried to quantify the level of methylation by cloning. The FCII PCR products were cloned and 10–12 colonies sequenced. The human NGFI-A core recognition motif, 5′-GCGGGGGCG-3′, includes two CpG sites (CpG41–42, Figure 4). Both of these positions were unmethylated in the majority of donors. In only 4 of the 26 donors less than 10% of the colonies screened were methylated at CpG41 (5′-end of the NGFI-A site). A similar level of methylation of CpG42 was found for only two donors (Figure 4). These results show that methylation of the NGFI-A site is possible in the human, but is neither uniform nor frequent. The level of methylation observed by cloning was in agreement with the low but detectable levels seen in the sequencing electropherogram.


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 promoter 1F. (A) In silico phylogenetic footprinting covers the end of exon 1B through to exon 1F. (B) Percentage methylation was measured by direct electropherogram reading (CpG1–30), or sequencing of 10–12 clones per donor (CpG31–43) after bisulphite sequencing of 26 donors, covering CpG 1–42. Methylation levels are expressed by colour, levels from the scale at the panel top. Blank squares indicate positions for which no data was obtainable. (C) CpG identifiers within the sequence of promoter 1F. (D) TRANSFAC prediction for promoter 1F. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2602793&req=5

Figure 4: In silico phylogenetic footprinting and bisulphite sequencing of promoter 1F. (A) In silico phylogenetic footprinting covers the end of exon 1B through to exon 1F. (B) Percentage methylation was measured by direct electropherogram reading (CpG1–30), or sequencing of 10–12 clones per donor (CpG31–43) after bisulphite sequencing of 26 donors, covering CpG 1–42. Methylation levels are expressed by colour, levels from the scale at the panel top. Blank squares indicate positions for which no data was obtainable. (C) CpG identifiers within the sequence of promoter 1F. (D) TRANSFAC prediction for promoter 1F. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.
Mentions: ISPF predicted for promoter 1F several high quality potential TFBS, including the NGFI-A-binding site reported before (25,35). Since methylation of the corresponding NGFI-A-binding site has been well documented in the rat, we tried to quantify the level of methylation by cloning. The FCII PCR products were cloned and 10–12 colonies sequenced. The human NGFI-A core recognition motif, 5′-GCGGGGGCG-3′, includes two CpG sites (CpG41–42, Figure 4). Both of these positions were unmethylated in the majority of donors. In only 4 of the 26 donors less than 10% of the colonies screened were methylated at CpG41 (5′-end of the NGFI-A site). A similar level of methylation of CpG42 was found for only two donors (Figure 4). These results show that methylation of the NGFI-A site is possible in the human, but is neither uniform nor frequent. The level of methylation observed by cloning was in agreement with the low but detectable levels seen in the sequencing electropherogram.

Bottom Line: 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.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.

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