Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2

Abstract

Background Exposure to drugs of abuse alters the epigenetic landscape of the brain’s reward regions such as the nucleus accumbens (NAc). We investigated how combinations of chromatin modifications affect genes that regulate responses to cocaine. We focused on autism-candidate 2 (Auts2), a gene linked to human evolution and cognitive disorders, which displays strong clustering of cocaine-induced chromatin modifications in this brain region. Methods We combined chromosome conformation capture (4C and 3C) and related approaches with behavioral paradigms relevant to cocaine phenotypes. Cell type-specific functions were assessed by FACS-sorting and viral-mediated overexpression in Cre-dependent mouse lines. Results We observed that Auts2 gene expression is increased by repeated cocaine administration specifically in D2-type medium spiny neurons (MSNs) in NAc, an effect seen in male but not female mice. Auts2 mRNA expression was also upregulated postmortem in NAc of male human cocaine addicts. We obtained evidence that chromosomal looping, bypassing 1,524 kb of linear genome, connects Auts2 to the calneuron 1 (Caln1) gene locus under baseline conditions. This looping was disrupted after repeated cocaine exposure, resulting in increased expression of both genes in D2 MSNs. Cocaine exposure reduces binding of CTCF, a chromosomal scaffolding protein, and increases histone and DNA methylation, at the Auts-Caln1 loop base in NAc. Cell type-specific overexpression of Auts2 or Calnl in D2 MSNs demonstrated that both genes promote cocaine reward. Conclusions These findings suggest that cocaine-induced alterations of neuronal 3D genome organization destabilize higher order chromatin at specific loci that regulate responses to the drug.

Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects

“…Using dCas9-SunTag proteins for targeting can even further increase on-target activity, because they are recruiting several effector domains to a target genomic locus, which was demonstrated with the DNMT3A catalytic domain [24,26,29] as well as with the TET1 catalytic domain used for DNA demethylation [29,30]. In the current study and for the first time, we combined the scFv-GCN4-DNMT3A-DNMT3L protein with the dCas9-SunTag system and compared its activity with the dCas9-DNMT3A-DNMT3L fusion protein published earlier [19,20]. Both systems demonstrated a strong on-target activity, with dCS being slightly more active (79% versus 84% DNA methylation).…”

“…The dC vector was cloned based on the previously published construct [19], in which EYFP was replaced by tagBFP using Gibson Assembly. The tagBFP sequence was obtained from Addgene plasmid #60903.…”

“…Inspired by this, an improved EpiEditor using a DNMT3A-DNMT3L single-chain fusion protein as an effector domain was developed [17]. It resulted in increased DNA methylation activity and was applied in several studies for gene silencing [17][18][19][20]. Another advancement was the introduction of dCas9 as a programmable DNA binding domain, which was used in several projects aiming for targeted DNA methylation [19][20][21][22][23][24][25][26][27].…”

How the epigenome integrates information and reshapes the synapse

“…Determining how these long-range interactions and the 3D architecture of the genome regulate neuronal gene expression, as recently investigated in several studies 33,231,232 , will be an important area of research.…”

Dairy cows – an opportunity in the research field of non-genetic inheritance?

“…Chromatin function can be altered by modifying DNA structure itself without affecting DNA sequence, for instance through DNA methylation [ 17 ]. Chromatin also contains a multitude of binding proteins, including histones or protamines as well as transcription and structural factors, which may be potential carriers of environmentally induced epigenetic changes in germ cells [ 18 ].…”