Background: FKBP51, encoded by the FKBP5 gene, is a key co-chaperone of the glucocorticoid receptor in response to stress that is linked to the development of severe psychiatric disorders. The FKBP5 gene codes four indexed alternative transcripts, and data from our laboratory suggests FKBP5 transcription might be regulated by DNA demethylation at key FKBP5 enhancers. It is likely that transcriptional and epigenetic regulation of the FKBP5 gene is tissue and cell-type specific. These patterns of regulation, that may be relevant for understanding disease risk, are yet to be determined in the human brain.
Methods: Postmortem brain samples from BA9 of 24 individuals without a history of neurological or psychiatric conditions were assessed. Ages of the subjects were evenly distributed from 37 to 88 years. Fresh frozen tissues were cryosectioned at 16μm and post-fixed in 4% PFA. For immunohistochemistry (IHC), samples were incubated with antibodies specific for a neuronal marker (TUJIII/MAP2), glia marker (GFAP) and full-length FKBP51 protein. For in situ hybridization (ISH), the RNAscope Multiplex kit was used to amplify probes targeting the FKBP5 long and short variants. Both IHC and ISH experiments were imaged with a Leica SP8 confocal microscope. An additional subset from BA18/19 of three control subjects were additionally FACS sorted into neuronal and non-neuronal subtypes with NeuN/DRAQ5 antibodies. DNA was extracted from NeuN+/- nuclei or bulk tissue, bisulfite converted and DNA methylation was assessed using a targeted bisulfite sequencing approach for the FKBP5 locus and the Infinium MethylationEPIC BeadChips.
Results: Our IHC data suggest that FKBP51 total protein expression might be limited to neuronal subtypes, although these data are undergoing validation. Similarly, ISH experiments show that FKBP5 long and short proteins are both expressed in BA9, with distributions in both nuclear and cytoplasmic compartments. These data are now being assessed for differences in levels and distributions of FKBP5 protein and transcripts in association with age. Total DNA methylation at FKBP5 regulatory CpGs (i.e. in promotor or enhancer regions) were significantly higher in neuronal populations compared to non-neuronal populations (P=0.011) and compared to bulk tissue (P=0.002). When assessing methylation at individual FKBP5 CpGs of interest, large differences in methylation were observed in CpGs close to regulatory regions of the FKBP5 gene, such as CTCF binding sites (e.g. cg06087101) and transcription start sites (e.g. cg01294490). These data are being confirmed in the larger BA9 cohort and with targeted bisulfite sequencing. Cell-type specific effects of methylation on transcription will also be assessed using qPCR.
Conclusions: These data provide initial evidence that the FKBP5 methylome and transcriptome are regulated in a cell-type specific manner in the human cortex. Further analyses are now being conducted to validate cell-type specific RNA and protein expression of FKBP5, and to determine associations of FKBP5 methylation and transcription. In addition, these results will be integrated with evidence from other brain cohorts to validate and determine if case-control differences exist in psychiatric disorders.