GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR- Cas9 -mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

32GABA type-A (GABA-A) receptors containing the α2 subunit (Gabra2) are expressed in most brain regions and are 33 critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, 34 affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is 35 modulated by sequence variants in several brain structures and populations, including F2 crosses originating from 36 C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global 37 reduction of Gabra2 brain mRNA and protein in the B6J strain relative to other inbred strains, and identify and validate 38 the causal mutation in B6J. The mutation is a single base pair intronic deletion located adjacent to a splice acceptor site 39 that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now 40 pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of 41 consomic lines. Repair of the deletion using CRISPR-Cas9 mediated gene editing on a B6J genetic background completely 42 restored brain levels of Gabra2 mRNA and protein. Comparison of transcript expression in hippocampus, cortex, and 43 striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in 44 striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or 45 inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal 46 function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic 47 studies in neurobiological research as this strain is widely used to generate genetically engineered mice and murine genetic 48 populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug 49

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