An expanded CAG repeat is the underlying genetic defect in Huntington disease, a disorder characterized by motor, psychiatric and cognitive deficits and striatal atrophy associated with neuronal loss. An accurate animal model of this disease is crucial for elucidation of the underlying natural history of the illness and also for testing experimental therapeutics. We established a new yeast artificial chromosome (YAC) mouse model of HD with the entire human HD gene containing 128 CAG repeats (YAC128) which develops motor abnormalities and age-dependent brain atrophy including cortical and striatal atrophy associated with striatal neuronal loss. YAC128 mice exhibit initial hyperactivity, followed by the onset of a motor deficit and finally hypokinesis. The motor deficit in the YAC128 mice is highly correlated with striatal neuronal loss, providing a structural correlate for the behavioral changes. The natural history of HD-related changes in the YAC128 mice has been defined, demonstrating the presence of huntingtin inclusions after the onset of behavior and neuropathological changes. The HD-related phenotypes of the YAC128 mice show phenotypic uniformity with low inter-animal variability present, which together with the age-dependent striatal neurodegeneration make it an ideal mouse model for the assessment of neuroprotective and other therapeutic interventions.

Huntingtin interacting protein 1 (HIP1) is a recently identi®ed component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1 ±/± ). HIP1 ±/± mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dosedependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1 ±/± mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor traf®cking in the central nervous system through its function in clathrin-mediated endocytosis.

The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.The 1-Mb imprinted domain on distal chromosome 7 (Chr 7) shares syntenic homology to the Beckwith-Wiedemann syndrome (BWS) region on human Chr 11p15.5 (69). Located less than 3 Mb from the telomere (Tel7q), this region contains two imprinting centers, IC1 and IC2. These conserved imprinting control elements are cis-acting sequences which carry opposite germ line DNA methylation marks and regulate the monoallelic expression of different flanking genes (9). In the proximal part of the Chr 7 domain, IC1 is located 2 kb upstream of the H19 promoter (Fig. 1A) (66). This sequence acquires a paternal DNA methylation imprint established during spermatogenesis and maintained throughout development (4,24,67). The methylated IC1 is required to initiate silencing of the paternal H19 allele (65), whereas the maternal IC1 controls the paternally expressed genes Igf2 and Ins2 via a methylation-sensitive insulator (6, 35). Distally, IC2 is located in intron 10 of Kcnq1 (Fig. 1A). This sequence is marked by a maternal DNA methylation imprint acquired during oogenesis (19). The unmethylated paternal IC2 is associated with the production of the Kcnq1ot1 noncoding RNA (ncRNA) and the silencing in cis of several genes in this subdomain (26). Consequently, these protein-coding genes are imprinted and expressed preferentially from the maternal Chr 7 homologue (62). These maternally expressed genes (MEGs) include transcripts expressed in the placenta and required for embryonic development, such as Ascl2 (31, 32), Cdkn1c (36), and Phlda2 (58). As in the case of IC1, IC2 appears to carry different allele-specific functions, such as promoter, enhancer, and CTCF binding insulator activities (2...

To study the substrate requirements for the histone 3'-end processing reaction of mammalian histone pre-mRNAs, we created a set of mutations in the sequences flanking the processing site of a mouse H3 gene. We found that deletion of the downstream purine-rich element hypothesized to interact with U7 small nuclear RNA abolishes in vitro 3'-end processing. Somewhat surprisingly, however, mutations in the hairpin loop element which destabilize or destroy the secondary structure reduce but do not abolish 3'-end processing. This is in apparent contrast to results obtained for the sea urchin system, where both sequence elements appear to be absolutely required for 3'-end formation.The 3' ends of eucaryotic mRNAs are generated by RNA processing rather than transcription termination (reviewed in references 4 and 23). The coordinately regulated histone mRNAs represent the major class of nonpolyadenylated RNAs in eucaryotic cells. Their 3' ends are formed by simple cleavage of a longer precursor RNA (3, 11. 14). Histone mRNAs differ from the majority of cellular RNAs not only by the absence of poly(A) tails, but in their general lack of introns. Thus, the typical histone pre-mRNA is subjected to a single RNA-processing reaction prior to its export to the cytoplasm, whereas most cellular mRNAs are processed by both splicing and polyadenylation.Levels of the coordinately regulated histone mRNAs are coupled to the cell cycle, apparently as a consequence of both transcriptional and posttranscriptional regulation (reviewed in reference 26). Since both pre-mRNA processing (3'-end formation) and mRNA stability have been implicated as important for the posttranscriptional regulation of histone mRNA abundance (reviewed in reference 19). a molecular description of the histone 3'-end processing reaction in mammalian cells will contribute significantly to our understanding of regulated RNA processing.Early work with sea urchin histone mRNAs showed that a small nuclear RNA (snRNA), U7 RNA, was required for 3'-end formation (8). This low-abundance snRNA was proposed to form intermolecular base pairs with two highly conserved sequence elements (28). Detailed mutagenesis studies (1-3, 9, 25) had determined that both of these elements, the hairpin loop lying just upstream and a conserved CAAGAAAGA sequence centered 13 to 14 nucleotides downstream of the cleavage site, were essential for generation of 3' ends on sea urchin histone pre-mRNAs. Direct support for base pairing between the 5' end of sea urchin U7 snRNA and the downstream element was provided by an experiment in which a processing defect in a sea urchin H3 gene (caused by a double base change in the CAAGAAAGA element) was suppressed by a U7 RNA, deletion of the entire hairpin loop in a mouse H4 substrate was shown to greatly reduce in vitro 3'-end processing (7). Thus, a large region containing both the hairpin loop and downstream consensus sequence has been delineated as important for correct 3' processing of mammalian histone pre-mRNAs, but detailed analyses determining the e...

To study the substrate requirements for the histone 3'-end processing reaction of mammalian histone pre-mRNAs, we created a set of mutations in the sequences flanking the processing site of a mouse H3 gene. We found that deletion of the downstream purine-rich element hypothesized to interact with U7 small nuclear RNA abolishes in vitro 3'-end processing. Somewhat surprisingly, however, mutations in the hairpin loop element which destabilize or destroy the secondary structure reduce but do not abolish 3'-end processing. This is in apparent contrast to results obtained for the sea urchin system, where both sequence elements appear to be absolutely required for 3'-end formation.