The epithelium-specific ETS protein EHF/ESE-3 is a context-dependent transcriptional repressor downstream of MAPK signaling cascades.

Exon trapping and cDNA selection procedures were used to search for novel genes at human chromosome 11p13, a region previously associated with loss of heterozygosity in epithelial carcinomas. Using these approaches, we found the ESE-2 and ESE-3 genes, coding for ETS domain-containing transcription factors. These genes lie in close proximity to the catalase gene within a approximately 200-kilobase genomic interval. ESE-3 mRNA is widely expressed in human tissues with high epithelial content, and immunohistochemical analysis with a newly generated monoclonal antibody revealed that ESE-3 is a nuclear protein expressed exclusively in differentiated epithelial cells and that it is absent in the epithelial carcinomas tested. In transient transfections, ESE-3 behaves as a repressor of the Ras- or phorbol ester-induced transcriptional activation of a subset of promoters that contain ETS and AP-1 binding sites. ESE-3-mediated repression is sequence- and context-dependent and depends both on the presence of high affinity ESE-3 binding sites in combination with AP-1 cis-elements and the arrangement of these sites within a given promoter. We propose that ESE-3 might be an important determinant in the control of epithelial differentiation, as a modulator of the nuclear response to mitogen-activated protein kinase signaling cascades.

Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein 1 gene.

The tub gene is a member of a small, well conserved neuronal gene family of unknown function. Mutations within this gene lead to early-onset blindness and deafness, as well as late-onset obesity and insulin resistance. To test the hypothesis that mutations within other members of this gene family would lead to similar phenotypes as observed in tubby mice, and hence have similar functional properties, we have generated null mutants of the tubby-like protein ( Tulp ) 1 gene by homologous recombination. Similarly to tubby mice, Tulp1 (-/-)mice exhibit an early-onset retinal degeneration with a progressive, rapid loss of photoreceptors, further supporting the notion that previously identified mutations within the human TULP1 gene are indeed causative of retinitis pigmentosa. However, in contrast to tubby mice, Tulp1 (-/-)mice exhibited normal hearing ability and, surprisingly, normal body weight despite the fact that both TUB and TULP1 are expressed in the same neurons within the hypothalamus in areas known to be involved in feeding behavior and energy homeo stasis. However, TUB and TULP1 show a distinctly different staining pattern in the nucleus of these neurons, perhaps explaining the difference in body weight between the Tulp1 (-/-)and tubby mutant mice.

Retinal degeneration in tulp1-/- mice: vesicular accumulation in the interphotoreceptor matrix.

PURPOSE: The Tulp1 gene is a member of the tubby gene family with unknown function. Mutations in the human TULP1 gene cause autosomal recessive retinitis pigmentosa. To understand the pathogenic mechanism associated with TULP1 mutations and to explore the physiologic function of this protein, we examined tissue distribution of the Tulp1 protein in normal mice and the photoreceptor disease phenotype in Tulp1-ablated mice. METHODS: Tissue distribution of the Tulp1 protein in normal mice was examined by immunoblotting and immunocytochemistry. The disease phenotype in tulp1-/- mice was studied by light and electron microscopy, electroretinography (ERG), and immunocytochemistry. These results were compared with another mouse model of retinal degeneration carrying a rhodopsin mutation. RESULTS: Tulp1 is found exclusively in photoreceptors, localizing predominantly in the inner segments. It is a soluble protein with an apparent molecular weight of approximately 70 kDa. Photoreceptor degeneration developed in tulp1-/- mice, with early involvement of both rods and cones. At the early stage of degeneration, rod and cone opsins, but not peripherin/RDS, exhibited prominent ectopic localization. Electron microscopy revealed massive accumulation of extracellular vesicles surrounding the distal inner segments. CONCLUSIONS: The function of Tulp1 is required to maintain viability of rod and cone photoreceptors. Extracellular vesicular accumulation is not a common phenomenon associated with photoreceptor degeneration but appears to be a distinct ultrastructural feature shared by a small group of retinal disease models. The defect in tulp1-/- mice may be consistent with a loss of polarized transport of nascent opsin to the outer segments.

Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse.

The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.

Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion that lengthens a glutamine segment in the novel huntingtin protein. To elucidate the molecular basis of HD, we extended the polyglutamine tract of the mouse homologue, Hdh, by targetted introduction of an expanded human HD CAG repeat, creating mutant HdhneoQ50 and HdhQ50 alleles that express reduced and wild-type levels of altered huntingtin, respectively. Mice homozygous for reduced levels displayed characteristic aberrant brain development and perinatal lethality, indicating a critical function for Hdh in neurogenesis. However, mice with normal levels of mutant huntingtin did not display these abnormalities, indicating that the expanded CAG repeat does not eliminate or detectably impair huntingtin's neurogenic function. Thus, the HD defect in man does not mimic complete or partial Hdh inactivation and appears to cause neurodegenerative disease by a gain-of-function mechanism.

Exon trapping and sequence-based methods of gene finding in transcript mapping of human 4p16.3.

We have applied exon amplification, GRAIL2 exon prediction and EST database searching to a 2 Mb segment of chromosome 4p16.3. Experimental and computational methods of identifying exons were comparable in efficiency and apparent false positive rate, but were complementary in gene identification, revealing distinct overlapping sets of expressed sequences. EST searching was most powerful when we considered only those ESTs that show evidence of splicing relative to the genomic sequence. The combination of the three gene finding methods produced a transcription map of 30 loci in this segment of 4p16.3 that includes known human genes, homologs of loci identified in rodents and several anonymous transcripts, including a putative novel DNA polymerase and a gene related to Drosophila ash1. While most of the genes in the region have been found, our data suggest that even with the entire DNA sequence available, complete saturation of the transcript map will require additional, focused experimental effort.

Inactivation of the mouse Huntington's disease gene homolog Hdh.

Huntington's disease (HD) is a dominant neurodegenerative disorder caused by expansion of a CAG repeat in the gene encoding huntingtin, a protein of unknown function. To distinguish between "loss of function" and "gain of function" models of HD, the murine HD homolog Hdh was inactivated by gene targeting. Mice heterozygous for Hdh inactivation were phenotypically normal, whereas homozygosity resulted in embryonic death. Homozygotes displayed abnormal gastrulation at embryonic day 7.5 and were resorbing by day 8.5. Thus, huntingtin is critical early in embryonic development, before the emergence of the nervous system. That Hdh inactivation does not mimic adult HD neuropathology suggests that the human disease involves a gain of function.

Normal and expanded Huntington's disease gene alleles produce distinguishable proteins due to translation across the CAG repeat.

BACKGROUND: An expanded CAG trinucleotide repeat is the genetic trigger of neuronal degeneration in Huntington's disease (HD), but its mode of action has yet to be discovered. The sequence of the HD gene places the CAG repeat near the 5' end in a region where it may be translated as a variable polyglutamine segment in the protein product, huntingtin. MATERIALS AND METHODS: Antisera directed at amino acid stretches predicted by the DNA sequence upstream and downstream of the CAG repeat were used in Western blot and immunohistochemical analyses to examine huntingtin expression from the normal and the HD allele in lymphoblastoid cells and postmortem brain tissue. RESULTS: CAG repeat segments of both normal and expanded HD alleles are indeed translated, as part of a discrete approximately 350-kD protein that is found primarily in the cytosol. The difference in the length of the N-terminal polyglutamine segment is sufficient to distinguish normal and HD huntingtin in a Western blot assay. CONCLUSIONS: The HD mutation does not eliminate expression of the HD gene but instead produces an altered protein with an expanded polyglutamine stretch near the N terminus. Thus, HD pathogenesis is probably triggered by an effect at the level of huntingtin protein.

Role of Rel-related factors in control of c-myc gene transcription in receptor-mediated apoptosis of the murine B cell WEHI 231 line.

Treatment of immature murine B lymphocytes with an antiserum against their surface immunoglobulin (sIg)M results in cell death via apoptosis. The WEHI 231 B cell line (IgM, kappa) has been used extensively as a model for this anti-Ig receptor-mediated apoptosis. Anti-sIg treatment of WEHI 231 cells causes an early, transient increase in the levels of c-myc messenger RNA and gene transcription, followed by a rapid decline below control values. Given the evidence for a role of the c-myc gene in promoting apoptosis, we have characterized the nature and kinetics of changes in the binding of Rel-related factors, which modulate c-myc promoter activity. In exponentially growing WEHI 231 cells, multiple Rel-related binding activities were detectable. The major binding species was identified as p50/c-Rel heterodimers; only minor amounts of nuclear factor kappa B (NF-kappa B) (p50/p65) were detectable. Cotransfection of an inhibitor of NF-kappa B (I kappa B)-alpha expression vector reduced c-myc-promoter/upstream/exon1-CAT reporter construct activity, indicating the role of Rel factor binding in c-myc basal expression in these cells. Treatment with anti-sIg resulted in a rapid transient increase in the rate of c-myc gene transcription and in the binding of Rel factors. At later times, formation of p50 homodimer complexes occurred. In cotransfection analysis, p65 and c-Rel expression potently and modestly transactivated the c-myc promoter, respectively, whereas, overexpression of the p50 subunit caused a significant drop in its activity. The role of activation of Rel-family binding was demonstrated directly upon addition of the antioxidant pyrrolidinedithiocarbamate, which inhibited the anti-sIg-mediated activation of the endogenous c-myc gene. Similarly, induction after anti-sIg treatment of a transfected c-myc promoter was abrogated upon cotransfection of an I kappa B-alpha expression vector. These results implicate the Rel-family in Ig receptor-mediated signals controlling the activation of c-myc gene transcription in WEHI 231 cells, and suggest a role for this family in apoptosis of this line, which is mediated through a c-myc signaling pathway.

Huntington's disease gene: regional and cellular expression in brain of normal and affected individuals.

Huntington's disease (HD) is an autosomal dominant disorder characterized by involuntary movements, dementia, and progressive, global, but regionally accentuated, brain atrophy. The disease affects the striatum most severely. An expansion of a trinucleotide repeat on chromosome 4p16.3 within the coding region of a gene termed IT15 has been identified as the mutation causing HD. The normal function of IT15 and the mechanisms by which the presence of the mutation causes HD are unknown. Although IT15 expression has been detected in the brain, as well as in other organ tissues, by Northern blot and in situ hybridization, it is not known whether a preferential regional or cellular expression of IT15 exists within the central nervous system of normal, affected, and presymptomatic individuals. Using quantitative in situ hybridization methods, we examined extensively the regional and cellular expression of IT15. In controls, IT15 expression was observed in all brain regions examined with the highest levels seen in cerebellum, hippocampus, cerebral cortex, substantia nigra pars compacta, and pontine nuclei. Expression in the striatum was intermediate and expression in the globus pallidus was low. IT15 was expressed predominantly in neurons; a low but significant level of expression was seen in glial cells. Analysis of grain counts per square micrometer in neurons showed that the regional differences in the level of mRNA expression were related to density and size of neurons in a given region and not primarily to differences in levels of mRNA expression in individual cells after correction for cell size. Neurons susceptible to degeneration in HD did not selectively express high levels of IT15 mRNA. In HD brains (grades 2-4), the distribution and levels of IT15 mRNA were comparable with controls in all areas except in neostriatum where the intensity of labeling was significantly reduced. Presymptomatic HD brains had a striatal expression similar to controls and surviving striatal neurons in more advanced HD had an expression of IT15 within normal limits. It is apparent from these results that the presence of expanded trinucleotide repeats in HD does not result in the absence of IT15 mRNA expression or in altered patterns or levels of expression. The lack of correlation between the levels of IT15 mRNA expression and susceptibility to degeneration in HD strongly suggests that the mutant gene acts in concert with other factors to cause the distinctive pattern of neurodegeneration in HD.

IT15 gene expression in fetal human brain.

To examine the expression of the gene which causes Huntington's disease (HD), IT15, during development, in situ hybridization of radiolabeled riboprobes was performed in human fetal (gestational ages 20-23 weeks) and adult brain. Optical densities of autoradiographs were determined in various brain regions and compared to cell density in those regions. IT15 expression was found in all regions of the fetal and adult brain, and there was a high degree of correlation of autoradiographic signal with cell number in all regions but germinal matrix in fetal brain and white matter in adult brain. These two regions are notable for their significant proportion of glial cells, and suggest that IT15 expression is predominantly neuronal. There was no preponderance of IT15 expression in striatal compartments in fetal brain as demonstrated by acetylcholinesterase activity, nor was there differential expression of IT15 in brain regions known to be particularly affected in HD. IT15 gene expression is present by 20 weeks gestation in human brain, and at that stage of development exhibits a pattern of distribution which is similar to adult brain. If a developmentally-regulated role for IT15 exists in the pathogenesis of HD, it must occur prior to 20 weeks gestation.

Synteny conservation of the Huntington's disease gene and surrounding loci on mouse Chromosome 5.

The mouse homologs of the Huntington's disease (HD) gene and 17 other human Chromosome (Chr) 4 loci (including six previously unmapped) were localized by use of an interspecific cross. All loci mapped in a continuous linkage group on mouse Chr 5, distal to En2 and I16, whose human counterparts are located on Chr 7. The relative order of the loci on human Chr 4 and mouse Chr 5 was maintained, except for a break between D5H4S115E and Idua/rd, with relocation of the latter to the opposite end of the map. The mouse HD homolog (Hdh) mapped within a cluster of seven genes that were completely linked in our data set. In human these loci span a approximately 1.8 Mb stretch of human 4p16.3 that has been entirely cloned. To date, there is no phenotypic correspondence between human and mouse mutations mapping to this region of synteny conservation.

Mouse Huntington's disease gene homolog (Hdh).

The incurable neurodegenerative disorder, Huntington's disease (HD), is caused by an expanded, unstable CAG repeat encoding a stretch of polyglutamine in a 4p16.3 gene (HD) of unknown function. Near the CAG repeat is a polyproline-encoding CCG repeat that shows more limited allelic variation. The mouse homologue, Hdh, has been mapped to chromosome 5, in a region devoid of mutations causing any comparable phenotype. We have isolated overlapping cDNAs from the Hdh gene and compared their sequences with the human transcript. The consensus mouse coding sequence is 86% identical to the human at the DNA level and 91% identical at the protein level. Despite the overall high level of conservation, Hdh possesses an imperfect CAG repeat encoding only seven consecutive glutamines, compared to the 13-36 residues that are normal in man. Although no evidence for polymorphic variation of the CAG repeat was seen, a nearby CCG repeat differed in length by one unit between several strains of laboratory mouse and Mus spretus. The absence of a long CAG repeat in the mouse is consistent with the lack of a spontaneous mouse model of HD. The information presented concerning the sequence of the mouse gene should facilitate attempts to create such a model.

Gametic but not somatic instability of CAG repeat length in Huntington's disease.

Instability of a CAG repeat in 4p16.3 has been found in Huntington's disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.

Molecular genetics of Huntington's disease.

Huntington's disease is an inherited disorder in which selective neuronal loss in the brain leads to a characteristic choreic movement disorder. The successful mapping of the Huntington's disease gene to chromosome 4 set off a torrent of similar studies in other inherited disorders as investigators attempted to locate and isolate human disease genes with this new approach. Although it took a decade-long quest since the initial mapping of the genetic defect, the gene causing Huntington's disease has recently been isolated. Discovery of the mutational mechanism causing Huntington's disease has explained some of the peculiarities of inheritance of this intriguing disorder and creates hope for a better understanding of the cause of neuronal cell death that could eventually lead to a treatment.

De novo expansion of a (CAG)n repeat in sporadic Huntington's disease.

Huntington's disease (HD) chromosomes contain an expanded unstable (CAG)n repeat in chromosome 4p16.3. We have examined nine families with potential de novo expression of the disease. With one exception, all of the affected individuals had 42 or more repeat units, well above the normal range. In four families, elderly unaffected relatives inherited the same chromosome as that containing the expanded repeat in the proband, but had repeat lengths of 34-38 units, spanning the gap between the normal and HD distributions. Thus, mutation to HD is usually associated with an expansion from an already large repeat.

Trinucleotide repeat length instability and age of onset in Huntington's disease.

The initial observation of an expanded and unstable trinucleotide repeat in the Huntington's disease gene has now been confirmed and extended in 150 independent Huntington's disease families. HD chromosomes contained 37-86 repeat units, whereas normal chromosomes displayed 11-34 repeats. The HD repeat length was inversely correlated with the age of onset of the disorder. The HD repeat was unstable in more than 80% of meiotic transmissions showing both increases and decreases in size with the largest increases occurring in paternal transmissions. The targeting of spermatogenesis as a particular source of repeat instability is reflected in the repeat distribution of HD sperm DNA. The analysis of the length and instability of individual repeats in members of these families has profound implications for presymptomatic diagnosis.