Project Summary: Most genetic variants associated with disease in genome-wide association studies (GWAS)
lie in non-coding gene regulatory elements (GRE; e.g., promoters and enhancers). GREs are tissue- and cell
type-specific and are identified through their epigenomic signatures, including low DNA methylation (DNAm),
DNA accessibility and certain histone modifications. The PsychENCODE Consortium has characterized brain
GREs across brain regions and developmental time points, as well as in the brains of psychiatric patients using
mostly DNA accessibility and histone modification marks. These marks, however, identify large regions of
enrichment (~300-2,000bp), providing only low resolution coverage of the important regulatory nucleotides, e.g.,
transcription factor binding sites. DNAm (especially cell type-specific DNAm) has received less attention,
although it has been linked to psychiatric disorders, including schizophrenia (SZ) and bipolar disorder (BD).
In the adult human brain, ~80% of CG and 1.5% of non-CG (CH) sites are methylated, and can be converted
to hydroxymethylcytosine (hmC) and further demethylated. In postmitotic neuronal genomes, mCH and hmC
accumulate to a significantly higher level than in other tissues--a distinct feature of the brain's epigenome.
Bisulfite sequencing (BS)-based approaches that are used to measure (h)mC can pinpoint GREs with single
base resolution, presenting a unique opportunity to refine the gene regulatory landscape of the brain cell types.
Here we aim to create reference DNAm maps [mC and hmC, using BS and oxidative (ox)BS sequencing]
and transcriptional profiles (using RNA-seq) in two major subtypes of neurons in the human dorsolateral
prefrontal cortex (DLPFC), namely excitatory glutamatergic (Glu) and inhibitory GABA-ergic neurons. The
proposed work is based on fluorescence-activated nuclear sorting (FANS) methods that we recently developed
to separate nuclei of different cell types from autopsied human brain, and on our recent findings that showed
unexpected relationships between DNA(h)m, GREs, and gene expression in the DLPFC Glu and GABA neurons.
We will perform these studies at key time points of postnatal brain development and adulthood to map
DNA(h)m within active neuron subtype-specific GREs that may be vulnerable to disruption during childhood and
adolescence periods that coincide with the critical processes of brain maturation and circuit refinement (Aim 1).
This work will be complemented with single nucleus mC profiling, which will allow us to define the developmental
trajectories of mC in discrete subpopulations of Glu and GABA neurons (Aim2).
Finally, we will profile Glu and GABA neurons in 150 autopsied DLPFC samples obtained from controls and
cases of SZ and BD, and will then map neuron subtype-specific gene expression and (hydroxy)methylation
quantitative trait loci (eQTL, mQTLS, hmQTLs) (Aim3). We will integrate QTL, transcriptome, and DNA(h)m data
with the results of SZ and BP GWAS to reveal DNA(h)m and gene expression-mediated causal risk variants and
genes, and to distinguish specific neuronal subtype(s) that are critical in the etiology of these disorders.
