[Show abstract][Hide abstract] ABSTRACT: Females with germline mutations in BRCA1 are predisposed to develop breast and ovarian cancers. A previous report indicated that BRCA1 colocalizes with and is necessary for the correct localization of XIST, a noncoding RNA that coats the inactive X chromosome (Xi) to mediate formation of facultative heterochromatin. A model emerged from this study suggesting that loss of BRCA1 in female cells could reactivate genes on the Xi through loss of the XIST RNA. However, our independent studies of BRCA1 and XIST RNA revealed little evidence to support this model. We report that BRCA1 is not enriched on XIST RNA-coated chromatin of the Xi. Neither mutation nor depletion of BRCA1 causes significant changes in XIST RNA localization or X-linked gene expression. Together, these results do not support a role for BRCA1 in promoting XIST RNA localization to the Xi or regulating XIST-dependent functions in maintaining the stability of facultative heterochromatin.
[Show abstract][Hide abstract] ABSTRACT: ELOVL4 was first identified as a disease-causing gene in Stargardt macular dystrophy (STGD3, MIM 600110.) To date, three ELOVL4 mutations have been identified, all of which result in truncated proteins which induce autosomal dominant juvenile macular degenerations. Based on sequence homology, ELOVL4 is thought to be another member within a family of proteins functioning in the elongation of long chain fatty acids. However, the normal function of ELOVL4 is unclear. We generated Elovl4 knockout mice to determine if Elovl4 loss affects retinal development or function. Here we show that Elovl4 knockout mice, while perinatal lethal, exhibit normal retinal development prior to death at day of birth. Further, postnatal retinal development in Elovl4 heterozygous mice appears normal. Therefore haploinsufficiency for wildtype ELOVL4 in autosomal dominant macular degeneration likely does not contribute to juvenile macular degeneration in STGD3 patients. We found, however, that Elovl4+/- mice exhibit enhanced ERG scotopic and photopic a and b waves relative to wildtype Elovl4+/+ mice suggesting that reduced Elovl4 levels may impact retinal electrophysiological responses.
Vision Research 04/2007; 47(5):714-22. DOI:10.1016/j.visres.2006.10.023 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with omega-hydroxy very long chain fatty acids (> or = C28) and accumulation of ceramides with non omega-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.
International journal of biological sciences 02/2007; 3(2):120-8. · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a screen for gene copy-number changes in mouse mammary tumors, we identified a tumor with a small 350-kb amplicon from a region that is syntenic to a much larger locus amplified in human cancers at chromosome 11q22. The mouse amplicon contains only one known gene, Yap, encoding the mammalian ortholog of Drosophila Yorkie (Yki), a downstream effector of the Hippo(Hpo)-Salvador(Sav)-Warts(Wts) signaling cascade, recently identified in flies as a critical regulator of cellular proliferation and apoptosis. In nontransformed mammary epithelial cells, overexpression of human YAP induces epithelial-to-mesenchymal transition, suppression of apoptosis, growth factor-independent proliferation, and anchorage-independent growth in soft agar. Together, these observations point to a potential oncogenic role for YAP in 11q22-amplified human cancers, and they suggest that this highly conserved signaling pathway identified in Drosophila regulates both cellular proliferation and apoptosis in mammalian epithelial cells.
Proceedings of the National Academy of Sciences 09/2006; 103(33):12405-10. DOI:10.1073/pnas.0605579103 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GADD45a is a transcription target of the breast tumor suppressor gene BRCA. It was recently shown that mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of Brca1 (Brca1(Delta11/Delta11)) or a Gadd45A-null mutation (Gadd45a(-/-)) suffer centrosome amplification. To study genetic interactions between these genes during centrosome duplication, we generated Brca1(Delta11/Delta)(11)Gadd45a(-/-) mice by crossing each mutant. We found that all Brca1(Delta11/Delta11)Gadd45a(-/-) embryos at embryonic days 9.5-10.5 were exencephalic and exhibited a high incidence of apoptosis accompanied by altered levels of BAX, BCL-2, and p53. The trigger for these events is likely the genetic instability arising from centrosome amplification that is associated, at least in part, with decreased expression of the NIMA-related kinase NEK2. We demonstrate that small interfering RNA-mediated suppression of Brca1 decreased Nek2 more dramatically in Gadd45a(-/-) cells than in wild-type cells and, conversely, that overexpression of Brca1 and/or Gadd45a up-regulated transcription of Nek2. Furthermore, we show that overexpression of Nek2 in Brca1-specific small interfering RNA-treated wild-type and Gadd45a(-/-) cells repressed abnormal centrosome amplification. These observations suggest that NEK2 plays a role in mediating the actions of BRCA1 and GADD45a in regulating centrosome duplication and in maintaining genetic stability.
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is a simple and powerful tool widely used for studying gene function in a number of species. Recently, inducible regulation of RNAi in mammalian cells using either tetracycline- or ecdysone-responsive systems has been developed to prevent potential lethality or non-physiological responses associated with persistent suppression of genes that are essential for cell survival or cell cycle progression. Here we show that the inducible regulation of RNAi also can be achieved by using a Cre-LoxP approach. We demonstrate that the insertion of a loxP-flanked neomycin cassette into RNA polymerase III promoter, which controls a vector-based RNAi unit, impairs the promoter activity. However, the expression of RNAi construct can be completely restored upon the removal of the neo cassette using a tamoxifen inducible Cre construct. We show that this system works with high efficiency in suppression of two endogenous genes, Fgfr2 and Survivin, in mouse embryonic stem (ES) cells, as evidenced by the decrease of levels of gene expression, reduced cell proliferation and colony formation. This system provides a potentially important yet simple approach to establish mutant mouse strains for functional study at defined stages upon turning on the inducible switches controlled by the Cre-LoxP system.
Nucleic Acids Research 02/2004; 32(10):e85. DOI:10.1093/nar/gnh083 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Smad4 is a central mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in mammary gland development and neoplasia, we disrupted this gene in mammary epithelium using a Cre-loxP approach. Smad4 is expressed in the mammary gland throughout development; however, its inactivation did not cause abnormal development of the gland during the first three pregnancies. Instead, lack of Smad4 gradually induced cell proliferation, alveolar hyperplasia and transdifferentiation of mammary epithelial cells into squamous epithelial cells. Consequently, all mutant mice developed squamous cell carcinoma and/or mammary abscesses between 5 and 16 months of age. We demonstrated that absence of Smad4 resulted in beta-catenin accumulation at onset and throughout the process of transdifferentiation, implicating beta-catenin, a key component of the Wnt signaling pathway, in the development of squamous metaplasia in Smad4-null mammary glands. We further demonstrated that TGFbeta1 treatment degraded beta-catenin and induced epithelial-mesenchymal transformation in cultured mammary epithelial cells. However, such actions were blocked in the absence of Smad4. These findings indicate that TGFbeta/Smad4 signals play a role in cell fate maintenance during mammary gland development and neoplasia.
Development 01/2004; 130(24):6143-53. DOI:10.1242/dev.00820 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast-cancer-associated gene 1 (BRCA1) is highly expressed in thymus and spleen. In this paper, we have studied lymphocyte development and tumorigenesis in mice carrying mutations in Brca1 and p53. We show that the deletion of Brca1 exon 11 (Brca1-delta11), which disrupts the full-length isoform, but not the short isoform of Brca1, does not interfere with lymphocyte development. This is true irrespective of p53 status, that is, whether it is wild type, heterozygous or homozygous for a null mutation. These data suggest that the expression of Brca1 short isoform alone is enough to maintain normal development of lymphocytes. However, it cannot suppress tumorigenesis as about 30% of Brca1(delta11/delta11)p53(+/-) mice develop thymic lymphoma between 3 and 7 months of age. We demonstrate that p53 plays an essential role in Brca1-associated lymphoma, as all the tumors from Brca1(delta11/delta11)p53(+/-) mice exhibit LOH of p53 and Brca1(delta11/delta11)p53(-/-) mice exhibited accelerated tumorigenesis. We further demonstrate that the Brca1-delta11 deficiency does not affect thymocyte proliferation; however, it increases genetic instability and triggers gamma-irradiation-induced apoptosis. The loss of p53 attenuates apoptosis and allows accumulation of further mutations in Brca1-delta11 thymocytes, eventually leading to thymic lymphoma formation.
[Show abstract][Hide abstract] ABSTRACT: Senescence may function as a two-edged sword that brings unexpected consequences to organisms. Here we provide evidence to support this theory by showing that the absence of the Brca1 full-length isoform causes senescence in mutant embryos and cultured cells as well as aging and tumorigenesis in adult mice. Haploid loss of p53 overcame embryonic senescence but failed to prevent the adult mutant mice from prematurely aging, which included decreased life span, reduced body fat deposition, osteoporosis, skin atrophy, and decreased wound healing. We further demonstrate that mutant cells that escaped senescence had undergone clonal selection for faster proliferation and extensive genetic/molecular alterations, including overexpression of cyclin D1 and cyclin A and loss of p53. These observations provide the first in vivo evidence that links cell senescence to aging due to impaired function of Brca1 at the expense of tumorigenesis.
Genes & Development 02/2003; 17(2):201-13. DOI:10.1101/gad.1050003 · 10.80 Impact Factor