Although studies of DNA methylation and its impact on chromatin architecture leave us with quite the buzz, this latest one adds a bit of cocaine to the mix to give it an extra kick. This stimulating study found cocaine use reduced DNA methylation at a key gene to change CTCF binding and alter 3D chromatin structure to ultimately regulate gene expression.
We’ve seen cocaine-related DNA demethylation at putative enhancers in the brain before. However, in this latest story, resourceful researchers in the lab of Gustavo Turecki (McGill University, Montreal, Canada) used reduced representation bisulfite sequencing (RRBS) to capture the genome-wide DNA methylation profile of cocaine dependence in human post-mortem brain (caudate) tissue. While there were many widespread changes, the team focused on Iroquois Homeobox (IRXA) gene family, finding:
- Some of the DMRs largest in size overlap with IRXA members IRX1 and IRX2
- There’s also increased gene expression (RNA-seq)
- Hypomethylation in exon 3 of IRX2 associates with chronic cocaine dependence in an independent cohort
- This change is specific to neuronal nuclei (not glial or epithelial cells), as detected via fluorescence-activated nuclei sorting (FACS)
- Irx2 exon 3 hypomethylation also occurs in mice that self-administer cocaine
By leveraging previous knowledge that the IRXA genes are regulated by enhancers in a 3D chromatin loop, these stimulating scientists hypothesized that the cocaine-related DNA methylation changes would impair the necessary CTCF binding and found a putative CTCF binding site near their hypomethylated DMR of interest in exon 3 of IRX2.
Next, they turned to human cell culture models to determine if DNA methylation can cause other molecular changes. A 3C (chromosome conformation capture) assay found IRX1 and IRX2 are physically closer than expected by chance, even more often in cells expressing both genes.
Experimentally increasing DNA methylation at the CTCF binding site by dCas9-based epigenome editing led to:
- Decreased IRX1 and IRX2 gene expression
- Decreased CTCF binding (detected by ChIP-qPCR)
- 3D chromatin interaction levels down to those expected by chance
With their human, mouse, and cell culture trifecta of data, the productive people behind this work are prepared to propose that cocaine dependence disrupts a methylation-sensitive CTCF binding site in exon 3 of IRX2.