, 2008). Our study supports the notion that mutant HDL2-CAG protein can perturb CBP-mediated transcription, in part, but not necessarily

exclusively, through the sequestration of CBP into NIs. Our current study does not rule out the possibility that mutant HDL2-CAG protein may also disrupt the function of other critical nuclear transcription factors such as TBP ( Rudnicki et al., 2008). Additional gene expression and epigenetic profiling, along with functional manipulation of these molecular pathways in HDL2 mice, will be necessary to critically evaluate their contribution in disease pathogenesis. Finally, our study reveals the complexity of disease pathogenesis mediated by trinucleotide repeat expansion. Together with Compound Library SCA8 (Moseley et al., 2006), our models provide another compelling example in which bidirectional transcription across an expanded CTG/CAG repeat leads to the expression of an antisense CAG transcript and previously unrecognized polyQ protein toxicity. Because the predicted HDL2-CAG protein has no known homology to any other protein in the human proteome beyond the polyQ stretch (data not shown), the function of this transcript and the small protein it encodes remains to be explored. Given the recent

discovery that antisense transcription is nearly ubiquitous throughout the mammalian genome (Katayama et al., 2005), our study highlights the BIBW2992 importance of examining antisense repeat-containing Cediranib (AZD2171) transcripts and their ORFs in the pathogenesis of other brain disorders. Human BAC (RP11-33A21) containing the JPH3 genomic locus from BACPAC Resource Center (Oakland Children’s Hospital, Oakland, CA) was engineered by using homologous recombination and microinjected into FvB/N embryos to generate the BAC transgenic mouse lines, BAC-HDL2, BAC-HDL2-STOP, and BAC-JPH3 (Yang et al., 1997 and Gong et al., 2002). These mouse lines were maintained in FvB/NJ inbred background. A second

BAC control mouse that was generated by using the wild-type JPH3 BAC (CTD-2195P9) was created and maintained in the C57/BL6 background (BAC-JPH3b6). More details about the transgene constructs and initial characterization of the mouse lines are in Supplemental Experimental Procedures. For RT-PCR analyses of JPH3 sense strand and antisense HDL2-CAG transcripts, total RNA was extracted by using the RNeasy Lipid mini-kit (QIAGEN, Valencia, CA). Synthesis of cDNA was primed by using either oligo(dT)20 (Invitrogen, Carlsbad, CA) or strand-specific oligonucleotide primers (see Table S1 for primers). Both 5′ and 3′ RACE analyses were performed by using FirstChoice® RLM-RACE kit (ABI) following the manufacturer’s instructions. A random-primed reverse transcription reaction and nested PCR was used to amplify 5′ and 3′ ends of the transcript (see Table S1). Quantitative RT-PCR analyses of BDNF transcripts in BAC-HDL2 and control cortices were performed by using published protocol ( Gray et al., 2008).