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

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.

Figure 1

Schematic representation of the Glucocorticoid receptor CpG island internal structure, the bisulphite sequencing strategy and primer locations. Known TFBS are shown as arrows and numbered from the ATG translation start codon in exon 2. § from (32), † from (53), ‡ homologous to the known rat site (25,27), ¶ the ephemeric exon 1G has only been detected in the rat as its homologue 1₈.

Figure 2.

In silico phylogenetic footprinting and bisulphite sequencing of promoter 1D. (A) In silico phylogenetic footprinting covers the start of the CpG island 340 nucleotides upstream of exon 1D through to exon 1D. (B) Percentage methylation was measured by direct electropherogram reading after bisulphite sequencing of 26 donors, covering CpG 6–27. Methylation levels are expressed by colour, levels from the scale at the panel top. (C) CpG identifiers and the unsequenced region of promoter 1D (grey background). (D) All TRANSFAC predicted human TFBS in promoter 1D are significant ISPF predictions.

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.

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 (CpG^1–30), or sequencing of 10–12 clones per donor (CpG^31–43) 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.

Figure 5.

In silico phylogenetic footprinting and bisulphite sequencing of promoter 1H. (A) In silico phylogenetic footprinting covers promoter 1H and part of exon 1H. (B) Percentage methylation was measured by direct electropherogram reading 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 1H and the unsequenced region (grey background). (D) TRANSFAC prediction for promoter 1H. Significant ISPF predictions are numbered corresponding to (A). Predictions in bold are high-quality ISPF predictions containing CpG dinucleotides.