PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain’s reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5—also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex—as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.National Institutes of Health (U.S.)National Institute on Drug AbuseIshibashi FoundationJapan Society for the Promotion of Science (KAKENHI 24591735)Japan Society for the Promotion of Science (KAKENHI 26290064)Japan Society for the Promotion of Science (KAKENHI 25116010

8-oxoguanine causes spontaneous de novo germline mutations in mice.

Spontaneous germline mutations generate genetic diversity in populations of sexually reproductive organisms, and are thus regarded as a driving force of evolution. However, the cause and mechanism remain unclear. 8-oxoguanine (8-oxoG) is a candidate molecule that causes germline mutations, because it makes DNA more prone to mutation and is constantly generated by reactive oxygen species in vivo. We show here that endogenous 8-oxoG caused de novo spontaneous and heritable G to T mutations in mice, which occurred at different stages in the germ cell lineage and were distributed throughout the chromosomes. Using exome analyses covering 40.9 Mb of mouse transcribed regions, we found increased frequencies of G to T mutations at a rate of 2 × 10(-7) mutations/base/generation in offspring of Mth1/Ogg1/Mutyh triple knockout (TOY-KO) mice, which accumulate 8-oxoG in the nuclear DNA of gonadal cells. The roles of MTH1, OGG1, and MUTYH are specific for the prevention of 8-oxoG-induced mutation, and 99% of the mutations observed in TOY-KO mice were G to T transversions caused by 8-oxoG; therefore, we concluded that 8-oxoG is a causative molecule for spontaneous and inheritable mutations of the germ lineage cells

Effects of Target Sequence and Sense versus Anti-sense Strands on Gene Correction with Single-stranded DNA Fragments

The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg–EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8–9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA

Targeted sequence alteration of a chromosomal locus in mouse liver

Targeted sequence alteration would be an attractive method in gene therapy and biotechnology. To achieve in vivo targeted sequence alteration, a tailed duplex DNA consisting of annealed 35mer and 794mer single-stranded DNAs was delivered by means of hydrodynamic tail vein injection into liver of transgenic mouse harboring a reporter gene (the rpsL gene) in its genome. The tailed DNA was designed for a conversion of ATC to AGC at codon 80 of the rpsL transgene. The anticipated T → G sequence alteration was induced in the transgene in the liver with an efficiency of ∼0.1%. These results demonstrate the significant potential of this method for applications in gene therapy and biotechnology

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

A 5-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches approximate to 330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment