Proteins with polyglutamine (polyQ) expansions accumulate in the nucleus and affect gene expression. The mechanism by which mutant huntingtin (htt) accumulates intranuclearly is not known; wild-type htt, a 350-kDa protein of unknown function, is normally found in the cytoplasm. N-terminal fragments of mutant htt, which contain a polyQ expansion (>37 glutamines), have no conserved nuclear localization sequences or nuclear export sequences but can accumulate in the nucleus and cause neurological problems in transgenic mice. Here we report that N-terminal htt shuttles between the cytoplasm and nucleus in a Ran GTPase-independent manner. Small N-terminal htt fragments interact with the nuclear pore protein translocated promoter region (Tpr), which is involved in nuclear export. PolyQ expansion and aggregation decrease this interaction and increase the nuclear accumulation of htt. Reducing the expression of Tpr by RNA interference or deletion of ten amino acids of N-terminal htt, which are essential for the interaction of htt with Tpr, increased the nuclear accumulation of htt. These results suggest that Tpr has a role in the nuclear export of N-terminal htt and that polyQ expansion reduces this nuclear export to cause the nuclear accumulation of htt.

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

Huntington disease (HD) is an adult-onset neurodegenerative disease caused by expansion of a polyglutamine (poly(Q) tract in the N-terminal region of huntingtin (htt). Although the precise mechanisms leading to neurodegeneration in HD have not been fully elucidated, transcriptional dysregulation has been implicated in disease pathogenesis. In HD, multiple N-terminal mutant htt fragments smaller than the first 500 amino acids have been found to accumulate in the nucleus and adversely affect gene transcription. It is unknown whether different htt fragments in the nucleus can differentially bind transcription factors and affect transcription. Here, we report that shorter N-terminal htt fragments, which are more prone to misfolding and aggregation, are more competent to bind Sp1 and inhibit its activity. These effects can be reversed by Hsp40, a molecular chaperone that reduces the misfolding of mutant htt. Our results provide insight into the beneficial effects of molecular chaperones and suggest that context dependent transcriptional dysregulation may contribute to differential toxicity of various N-terminal htt fragments. Huntington disease (HD)3 is an autosomal dominant neurodegenerative disorder resulting from expansion (Ͼ37 repeats) of a polyglutamine (poly(Q)) repeat in the N-terminal region of huntingtin (htt), a 350-kDa protein of unknown function. Proteolytic cleavage of full-length htt, which is predominantly cytoplasmic, generates N-terminal htt fragments that accumulate abnormally and form inclusions over time in neuronal nuclei (1). The nucleus is thought to be a primary site of poly(Q) toxicity, as blocking the nuclear entry of htt suppresses its ability to cause cell death (2), whereas targeting htt to the nucleus by the addition of a nuclear localization signal (NLS) causes a more severe phenotype (3-5).In the nucleus, mutant htt abnormally interacts with a number of transcription factors (6). Accordingly, the nuclear pathology observed in HD is thought to be largely due to transcriptional dysregulation (7,8). In fact, mRNA levels are altered for specific genes in HD mouse and cell models as well as in post-mortem human HD brain (7-11). Mutant htt has a higher affinity than normal htt for certain transcription factors, such as Sp1 (12-15), and these aberrant interactions can functionally deactivate transcription factors by titrating them away from their normal DNA binding sites (12)(13)(14)(15)(16).Biochemical analysis of HD knock-in mice that express a 150-glutamine repeat in the endogenous mouse htt (17) revealed the presence of multiple N-terminal htt fragments smaller than the first 508 amino acids in the nucleus (18), consistent with the notion that cleavage of mutant htt is a key event in HD pathology (19,20). Because htt protein length can influence its cellular toxicity and ability to cause neurodegeneration in HD cellular models and transgenic mice (21, 22), it is important to know whether nuclear N-terminal htt fragments of different length can differentially affect gene transcription. Und...

Genotyping mice by DNA based methods is both laborious and costly. As an alternative, we systematically examined fluorescent proteins expressed in the lens as transgenic markers for mice. A set of eye markers has been selected such that double and triple transgenic animals can be visually identified and that fluorescence intensity in the eyes can be used to distinguish heterozygous from homozygous mice. Taken together, these eye markers dramatically reduce the time and cost of genotyping transgenics and empower analysis of genetic interaction.