The 5′ untranslated region (UTR) of the glucocorticoid receptor (GR) plays a key role in determining tissue-specific expression and protein isoforms. Analysis of the 5′ UTR of the human GR (hGR) has revealed 11 splice variants of the hGR exon 1, based on seven exon 1s, four of which (1-D to 1-F and 1-H) were previously unknown. All of the exon 1 variants have unique splice donor sites and share a common exon 2 splice acceptor site. Due to an upstream in-frame TGA stop codon the predicted translation from all splice variants is identical. The four new exon 1s show remarkable similarity with their rat homologues. Exon 1-D starts and finishes 17 and 36 bp upstream of the corresponding ends of the rat exon 1₄. Exon 1-E is only 6 bp longer than its homologue exon 1₅. Exon 1-F contains two short inserts of 11 and 6 bp when compared with the rat 1₇. 1-H is 18 bp longer than the corresponding rat 1₁₁. In addition to these new exons, we found that the human exon 1-C occurs as three distinct splice variants, covering the region homologous to the rat exons 1₉ and 1₁₀. All of the alternative hGR exons 1s presented here were found to be transcribed in human tissue. The human hippocampus expresses mRNA of all the exon 1 variants, while the expression of the other exon 1s seems to be tissue specific. While exon 1-D is only in the hippocampus, exons 1-E and 1-F are also detected in the immune system, and exon 1-H additionally in the liver, lung and smooth muscle. The 5′ region of the hGR is more complex than previously thought, and we suggest that each of these untranslated first exons have a distinct proximal promoter region, providing additional depth to the mechanisms available for tissue-specific expression of the hGR isoforms.

DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC), is considered to be one of the principal interfaces between the genome and our environment, and it helps explain phenotypic variations in human populations. Initial reports of large differences in methylation level in genomic regulatory regions, coupled with clear gene expression data in both imprinted genes and malignant diseases, provided easily dissected molecular mechanisms for switching genes on or off. However, a more subtle process is becoming evident, where small (<10 %) changes to intermediate methylation levels are associated with complex disease phenotypes. This has resulted in two clear methylation paradigms. The latter “subtle change” paradigm is rapidly becoming the epigenetic hallmark of complex disease phenotypes, although we are currently hampered by a lack of data addressing the true biological significance and meaning of these small differences. Our initial expectation of rapidly identifying mechanisms linking environmental exposure to a disease phenotype led to numerous observational/association studies being performed. Although this expectation remains unmet, there is now a growing body of literature on specific genes, suggesting wide ranging transcriptional and translational consequences of such subtle methylation changes. Data from the glucocorticoid receptor (NR3C1) has shown that a complex interplay between DNA methylation, extensive 5′UTR splicing, and microvariability gives rise to the overall level and relative distribution of total and N-terminal protein isoforms generated. Additionally, the presence of multiple AUG translation initiation codons throughout the complete, processed mRNA enables translation variability, hereby enhancing the translational isoforms and the resulting protein isoform diversity, providing a clear link between small changes in DNA methylation and significant changes in protein isoforms and cellular locations. Methylation changes in the NR3C1 CpG island alters the NR3C1 transcription and eventually protein isoforms in the tissues, resulting in subtle but visible physiological variability. This review addresses the current pathophysiological and clinical associations of such characteristically small DNA methylation changes, the ever-growing roles of DNA methylation and the evidence available, particularly from the glucocorticoid receptor of the cascade of events initiated by such subtle methylation changes, as well as addressing the underlying question as to what represents a genuine biologically significant difference in methylation.

Endogenous opioid peptides and prescription opioid drugs modulate pain, anxiety and stress by activating opioid receptors, currently classified into four subtypes. Here we demonstrate that ACKR3/CXCR7, hitherto known as an atypical scavenger receptor for chemokines, is a broad-spectrum scavenger of opioid peptides. Phylogenetically, ACKR3 is intermediate between chemokine and opioid receptors and is present in various brain regions together with classical opioid receptors. Functionally, ACKR3 is a scavenger receptor for a wide variety of opioid peptides, especially enkephalins and dynorphins, reducing their availability for the classical opioid receptors. ACKR3 is not modulated by prescription opioids, but we show that an ACKR3-selective subnanomolar competitor peptide, LIH383, can restrain ACKR3’s negative regulatory function on opioid peptides in rat brain and potentiate their activity towards classical receptors, which may open alternative therapeutic avenues for opioid-related disorders. Altogether, our results reveal that ACKR3 is an atypical opioid receptor with cross-family ligand selectivity.