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X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containing TAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 of TAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression of TAF1 and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells, XDP fibroblasts exhibited decreased expression of TAF1 transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34′), was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration., Summary: This study describes a new iPSC model of X-linked dystonia-parkinsonism (XDP), which was initially validated by demonstrating a similar transcriptional defect as has been previously reported in XDP brain tissue.

Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG) deletion that results in the loss of a glutamic acid residue (ΔE302/303) in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH) proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine.Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4) levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the wild type and the mutant torsin proteins.

SUMMARY A previously unrecognized mechanism through which large ribonucleoprotein (megaRNP) granules exit the nucleus is by budding through the nuclear envelope (NE). This mechanism is akin to the nuclear egress of herpes-type viruses and is essential for proper synapse development. However, the molecular machinery required to remodel the NE during this process is unknown. Here, we identify Torsin, an AAA-ATPase that in humans is linked to dystonia, as a major mediator of primary megaRNP envelopment during NE budding. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the messenger RNAs contained within fail to reach synaptic sites, preventing normal synaptic protein synthesis and thus proper synaptic bouton development. These studies begin to establish the cellular machinery underlying the exit of megaRNPs via budding, offer an explanation for the ‘‘nuclear blebbing’’ phenotype found in dystonia models, and provide an important link between Torsin and the synaptic phenotypes observed in dystonia.

Most eukaryotic cells abundantly express polypeptide chain elongation factor-1 alpha (EF-1 alpha), an enzyme which catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes. In this study, a series of deletion and scanning mutations was introduced in the promoter region of human EF-1 alpha chromosomal gene. Mutated promoters were fused to the bacterial chloramphenicol acetyltransferase gene, and their promoter activity was determined in human HeLa cells. These analyses indicated that both the 5'-flanking region and the first intron of the EF-1 alpha gene are essential for its promoter activity. The region responsible in the intron contains several Sp1 and Ap1 elements which seem to have additive effects on its promoter activity. In the 5'-flanking region, two cis-elements (EFP1 and EFP2) which work interdependently were identified. Gel shift assay with EFP1 and EFP2 elements indicated that several nuclear factors bind to EFP1 and EFP2, and one of the three retarded bands with EFP2 could be super-shifted with the anti-Sp1 antibody. These results indicate that Sp1 or its related factor cooperatively enhances the expression of the EF-1 alpha gene in the 5' flanking region.

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.

A new computational method uses gene expression databases and transcription factor binding specificities to describe regulatory elements in the Drosophila A/P patterning network in unprecedented detail., Cis-regulatory modules that drive precise spatial-temporal patterns of gene expression are central to the process of metazoan development. We describe a new computational strategy to annotate genomic sequences based on their “pattern generating potential” and to produce quantitative descriptions of transcriptional regulatory networks at the level of individual protein-module interactions. We use this approach to convert the qualitative understanding of interactions that regulate Drosophila segmentation into a network model in which a confidence value is associated with each transcription factor-module interaction. Sequence information from multiple Drosophila species is integrated with transcription factor binding specificities to determine conserved binding site frequencies across the genome. These binding site profiles are combined with transcription factor expression information to create a model to predict module activity patterns. This model is used to scan genomic sequences for the potential to generate all or part of the expression pattern of a nearby gene, obtained from available gene expression databases. Interactions between individual transcription factors and modules are inferred by a statistical method to quantify a factor's contribution to the module's pattern generating potential. We use these pattern generating potentials to systematically describe the location and function of known and novel cis-regulatory modules in the segmentation network, identifying many examples of modules predicted to have overlapping expression activities. Surprisingly, conserved transcription factor binding site frequencies were as effective as experimental measurements of occupancy in predicting module expression patterns or factor-module interactions. Thus, unlike previous module prediction methods, this method predicts not only the location of modules but also their spatial activity pattern and the factors that directly determine this pattern. As databases of transcription factor specificities and in vivo gene expression patterns grow, analysis of pattern generating potentials provides a general method to decode transcriptional regulatory sequences and networks., Author Summary The developmental program specifying segmentation along the anterior-posterior axis of the Drosophila embryo is one of the best studied examples of transcriptional regulatory networks. Previous work has identified the location and function of dozens of DNA segments called cis-regulatory “modules” that regulate several genes in precise spatial patterns in the early embryo. In many cases, transcription factors that interact with such modules have also been identified. We present a novel computational framework that turns a qualitative and fragmented understanding of modules and factor-module interactions into a quantitative, systems-level view. The formalism utilizes experimentally characterized binding specificities of transcription factors and gene expression patterns to describe how multiple transcription factors (working as activators or repressors) act together in a module to determine its regulatory activity. This formalism can explain the expression patterns of known modules, infer factor-module interactions and quantify the potential of an arbitrary DNA segment to drive a gene's expression. We have also employed databases of gene expression patterns to find novel modules of the regulatory network. As databases of binding motifs and gene expression patterns grow, this new approach provides a general method to decode transcriptional regulatory sequences and networks.

All muscle cells develop from the mesoderm, which is the middle germ layer in the early embryo. The mesoderm itself derives from the ventral cells of the blastoderm stage embryo. Therefore, the regulatory events controlling dorsal-ventral development in the oocyte and the early embryo are the earliest events in muscle formation. The first stage in dorsal-ventral development can be traced back to the oocyte, where Gurken-Torpedo signaling establishes dorsal-ventral asymmetry. The ventral half of the oocyte is then allowed to express Pipe, which serves to activate a series of serine proteases. These activation events ultimately lead to the stimulation of the Toll receptor in the ventral side of the early embryo. In the final stage of this Toll maternal cascade there is the formation of the Dorsal protein gradient in the ventral nuclei of the embryo blastoderm. The Dorsal gradient both activates and represses zygotic gene expression to establish mesodermal cell fate and promote mesoderm invagination. The invaginated mesoderm then differentiates into appropriate muscle tissue types according to further positional information.

Granulocyte colony-stimulating factor (G-CSF) is produced from macrophages in response to lipopolysaccharide (LPS). GPE1, a cis-acting element of the G-CSF gene promoter, functioned as an LPS-inducible element. We isolated cDNA and a chromosomal gene encoding the mouse GPEI-binding protein (GPE1-BP). A 150-amino acid protein deduced from the cDNA has a basic domain and a leucine zipper motif, and seems to be identical to that of recently isolated Ig/EBP. The nuclear extract from COS cells transfected with the cDNA showed GPE1-binding activity. Transcripts were ubiquitously detected, and may be spliced from two exons of a single gene.

The Drosophila fusilli (fus) gene was identified in a genetic screen for dominant maternal enhancers of an unusual dorsalizing mutation in the cactus gene, cactE10. While females that are heterozygous for the cactE10 allele produce embryos with wild-type dorsal–ventral patterning, more than 90% of the embryos produced by females that are heterozygous for both cactE10 and the fus1 mutation are weakly dorsalized. Loss offusilli activity causes lethality during embryogenesis but not dorsal–ventral patterning defects, indicating that fusilli is important in more than one developmental process. The fusilli gene encodes a protein with RNA binding motifs related to those in mammalian hnRNP F and H, which play roles in regulated RNA splicing. The fusilli RNA is not present in the oocyte or early embryo, and germ-line clones of fusilli mutations have no maternal effect on dorsal–ventral patterning, indicating that the fusilli maternal effect does not depend on germ-line expression of the gene. Because the fusilli RNA is present in ovarian follicle cells, we propose that fusilli acts downstream of the Drosophila EGF receptor to control the biogenesis of follicle cell transcripts that control the initial dorsal–ventral asymmetry of the embryo.