Epigenetic and Transcriptional Control of the Opioid Prodynorphine Gene: In-Depth Analysis in the Human Brain

Abstract

Neuropeptides serve as neurohormones and local paracrine regulators that control neural networks regulating behavior, endocrine system and sensorimotor functions. Their expression is characterized by exceptionally restricted profiles. Circuit-specific and adaptive expression of neuropeptide genes may be defined by transcriptional and epigenetic mechanisms controlled by cell type and subtype sequence-specific transcription factors, insulators and silencers. The opioid peptide dynorphins play a critical role in neurological and psychiatric disorders, pain processing and stress, while their mutations cause profound neurodegeneration in the human brain. In this review, we focus on the prodynorphin gene as a model for the in-depth epigenetic and transcriptional analysis of expression of the neuropeptide genes. Prodynorphin studies may provide a framework for analysis of mechanisms relevant for regulation of neuropeptide genes in normal and pathological human brain.

Keywords: epigenetics; human brain; prodynorphin; transcription.

Figure 1

Human PDYN gene (modified screenshot from UCSC Genome Browser). (a) Gene structure. (b) Promoter PDYN region with VNTR and TSSs. Conservation across vertebrates. (c) Canonical PDYN mRNAs and transcripts initiated in exon 4. Their conservation across vertebrates. Non-coding sequences are shown by thin dark blue line; coding sequences by thick dark blue line; dynorphin peptides-encoding sequences by yellow. CN, caudate nucleus; NAc, nucleus accumbens; Put, putamen. Modified from [41].

Figure 2

PDYN mRNAs coding for the full-length (FL, (a)) and truncated proteins (b). (a) Transcripts encoding FL-PDYN protein. The dominant FL1-PDYN and shorter transcripts including FL2-PDYN and GTEx1-3 and testis-specific Taf I and Taf II transcripts differ in the first and second exons, and in TSS. (b) PDYN mRNAs encoding truncated PDYN proteins including alternatively spliced Sp1, Sp2, ΔSP-PDYN and ΔSP/NLS-PDYN transcripts, and transcripts initiated within the coding part of exon 4 (T1 and T2). Signal peptide is truncated in both ΔSP- and ΔSP/NLS-PDYN. Putative nuclear localization signal (NLS) is located in the dynorphin domain. Curved arrows show initiation of translation. Modified from [46].

Figure 3

Structure of ΔSP-PDYN mRNA and protein, PDYN pathogenic mutations causing SCA23, and nuclear localization of ∆SP-PDYN protein. (a) ΔSP-PDYN encode ΔSP-PDYN protein with truncated signal peptide. Sequences of opioid peptides α-neoendorphin (α-NE), dynorphin A (Dyn A), dynorphinB (Dyn B), and big dynorphin (Big Dyn) are shown in yellow. Pathogenic mutations form a mutational hot spot that is localized within the pathogenic big dynorphin sequence with dynorphin A as a core. (b,c) PDYN immunoreactivity (red) in the nuclei (green) of neurons in the human caudate nucleus. (d) Double labeling (yellow) of neuronal nuclei (arrows) in 3D confocal reconstruction projections. Scale bar, 20 μm. Modified from [46].

Figure 4

Locus of human PDYN with targets for transcription factors. (a) Genomic organization showing PDYN, the antisense AK090681 transcript and transcription factor targets deposited on UCSC Genome Browser. (b) Verified and putative transcription factor binding elements, promoter VNTR, CpG islands 1 and 2 (CGI 1 and CGI 2), PDYN pathogenic mutations causing neurodegeneration, and DNase I hypersensitivity sequence (DHS), and CpG-SNPs association with alcoholism. Thin light blue line shows non-coding RNA, thick dark blue line coding region, vertical yellow lines dynorphin sequences. Modified from [41].

Figure 5

Model for epigenetic and transcriptional regulation of neuronal PDYN transcription. In neurons, USF2 binds to E-box in the promoter CGI that is hypomethylated and enriched in 5-hydroxymethylcytosine (5-hmC). In glia, the CGI is hypermethylated. DMR2 and DMR1/CGI exhibit methylation patterns that are opposite between them and between neurons and glia for each of them. In non-neuronal cells, DMR2 may be targeted by methylation-sensitive transcriptional repressor such as DREAM, while in neurons by a methylation-dependent transcriptional activator. In glia, the DMR1/CGI may be wrapped in a nucleosome, that prevents transcriptional initiation. These mechanisms may underlie contrasting PDYN expression in neurons and glia. Modified from [79].

Figure 6

The CpG-SNP hypothesis. (a) Genetic, epigenetic and environmental factors are mechanistically integrated at CpG-SNPs that may be methylated and hydroxymethylated at the C-allele. Two alleles and three cytosine epialleles may differentially affect gene transcription and thereby differently contribute to deasease predisposition [89]. (b) PDYN SNPs variants associated with alcoholism are shown in blue while those forming CpGs in red. (c) T-allele-binding factor (Ta-BF) has high affinity for the T and methylated C alleles of the 3′-UTR CpG-SNP but not to unmethylated C allele. The high affinity interaction may be a basis for transcriptional activation by this DNA-binding protein.

Figure 7

Correlation of USF2 and PDYN (a,b), and their co-localization (c–e) in the human dlPFC. (a,b) The estimated effect with 95% confidence interval. Immunoreactivity of (c) PDYN, and (d) USF2 in the cytoplasm and nuclei of the layer V neurons, respectively. (e) Double labeling of PDYN and USF2 in the same neuron. Scale bars, 50 μm (c,d); and 25 μm (e). Modified from [79].