Biomedical subjects

Z D Sharp

Publications and source records attributed to Z D Sharp.

At least 19 recordsLinked to original sources

PubMed2000

Subnuclear dynamics and transcription factor function.

At a simplistic level, the nucleus can be thought of as singular organelle with a nuclear envelope designed to isolate the biochemical reactions required for gene transcription and DNA replication from the cytoplasm. It has become increasingly clear, however, that many higher levels of organization exist within the nucleus. A functional consequence of this organization is that nuclear processes that include transcription, RNA processing, and DNA synthesis are isolated to specific intranuclear domains to ensure efficiency. With the advent of GFP technologies and increasingly sophisticated instrumentation, we have continued to dissect the relationship between organization and function, in particular using live cells and ligand-dependent steroid receptors as a model system. These new opportunities have provided further insight into receptor function and the dependence upon intranuclear dynamics that take place within minutes of hormone addition. J. Cell. Biochem. Suppl. 35:99-106, 2000.

Animals↗

PubMed2000

Chuar Group of the Grand Canyon: record of breakup of Rodinia, associated change in the global carbon cycle, and ecosystem expansion by 740 Ma.

The Chuar Group (approximately 1600 m thick) preserves a record of extensional tectonism, ocean-chemistry fluctuations, and biological diversification during the late Neoproterozoic Era. An ash layer from the top of the section has a U-Pb zircon age of 742 +/- 6 Ma. The Chuar Group was deposited at low latitudes during extension on the north-trending Butte fault system and is inferred to record rifting during the breakup of Rodinia. Shallow-marine deposition is documented by tide- and wave-generated sedimentary structures, facies associations, and fossils. C isotopes in organic carbon show large stratigraphic variations, apparently recording incipient stages of the marked C isotopic fluctuations that characterize later Neoproterozoic time. Upper Chuar rocks preserve a rich biota that includes not only cyanobacteria and algae, but also heterotrophic protists that document increased food web complexity in Neoproterozoic ecosystems. The Chuar Group thus provides a well-dated, high-resolution record of early events in the sequence of linked tectonic, biogeochemical, environmental, and biological changes that collectively ushered in the Phanerozoic Eon.

Animals↗

PubMed1999

Expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G(2)/M checkpoint control.

BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA damage. BRCA2 binds to Rad51 through its BRC repeats. In support of the biological significance of this interaction, we found that the complex of BRCA2 and Rad51 in breast cancer MCF-7 cells was diminished upon conditional expression of a wild-type, but not a mutated, BRC4 repeat using the tetracycline-inducible system. Cells expressing a wild-type BRC4 repeat showed hypersensitivity to gamma-irradiation, an inability to form Rad51 radiation-induced foci, and a failure of radiation-induced G(2)/M, but not G(1)/S, checkpoint control. These results strongly suggest that the interaction between BRCA2 and Rad51 mediated by BRC repeats is critical for the cellular response to DNA damage.

Amino Acid Sequence↗

PubMed1999

Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response.

BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation, BRCA1 was detected in discrete foci in the nucleus, which colocalize with hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50, hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells carrying a homozygous mutation in BRCA1 but was restored by transfection of wild-type BRCA1. Ectopic expression of wild-type, but not mutated, BRCA1 in these cells rendered them less sensitive to the DNA damage agent, methyl methanesulfonate. These data suggest that BRCA1 is important for the cellular responses to DNA damage that are mediated by the hRad50-hMre11-p95 complex.

Acid Anhydride Hydrolases↗

PubMed1999

Binding of CtIP to the BRCT repeats of BRCA1 involved in the transcription regulation of p21 is disrupted upon DNA damage.

Mutations in BRCA1 are responsible for nearly all of the hereditary ovarian and breast cancers, and about half of those in breast cancer-only kindreds. The ability of BRCA1 to transactivate the p21 promoter can be inactivated by mutation of the conserved BRCA1 C-terminal (BRCT) repeats. To explore the mechanisms of this BRCA1 function, the BRCT repeats were used as bait in a yeast two-hybrid screen. A known protein, CtIP, a co-repressor with CtBP, was found. CtIP interacts specifically with the BRCT repeats of BRCA1, both in vitro and in vivo, and tumor-derived mutations in this region abolished these interactions. The association of BRCA1 with CtIP was also abrogated in cells treated with DNA-damaging agents including UV, gamma-irradiation, and adriamycin, a response correlated with BRCA1 phosphorylation. The transactivation of the p21 promoter by BRCA1 was diminished by expression of exogenous CtIP and CtBP. These results suggest that the binding of the BRCT repeats of BRCA1 to CtIP/CtBP is critical in mediating transcriptional regulation of p21 in response to DNA damage.

BRCA1 Protein↗

PubMed1999

Thyroid hormone, T3-dependent phosphorylation and translocation of Trip230 from the Golgi complex to the nucleus.

Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by the retinoblastoma tumor-suppressor protein. In an examination of its subcellular distribution, Trip230 localized predominantly to the vicinity of the Golgi instead of the nucleus, as other nuclear hormone receptor coactivators. Using a series of deletion mutants, a critical region identified for Golgi area targeting coincided with a previously defined thyroid hormone receptor-binding domain of Trip230. During cell cycle progression, the expression level of Trip230 is constant and a significant portion is imported into the nucleus at S phase. Within an hour of treating cells with T3, Trip230 immunofluorescence transiently colocalized with TR in prominent subnuclear structures. T3-dependent nuclear import of Trip230 does not require new protein synthesis. Coincident with T3 treatment and nuclear import, newly phosphorylated residue(s) appeared in Trip230, suggesting that phosphorylation may be involved in its nuclear import. These findings provided a novel mechanism for the regulation of nuclear hormone transcription factors by hormone-responsive phosphorylation and nuclear import of cytoplasmically located coactivators.

Animals↗

PubMed1999

Subnuclear partitioning and functional regulation of the Pit-1 transcription factor.

Subnuclear compartmentation is postulated to play an important role in many aspects of nuclear metabolism. To directly test an application of this model to transcription factor function, we examined the subnuclear partitioning behavior of Pit-1, a tissue-specific, POU-class transactivator. Biochemical and in situ assays indicate the nuclear pool of Pit-1 is normally divided between two compartments: the majority being differentially soluble in detergent, and a significant insoluble fraction (approximately 20%) bound to the nuclear matrix. Examination of Pit-1 deletion mutants and chimeric fusions reveal the highly conserved 66 amino acid POU-specific domain contains a necessary and sufficient nuclear matrix targeting signal. The nuclear partitioning behavior of several natural or engineered point mutations of Pit-1 was also examined. Surprisingly, the inactive point mutants were completely matrix-bound, irrespective of their ability to bind Pit-1 specific DNA. These results suggest that dynamic partitioning of Pit-1 is a component of its normal transactivator function that takes place upon the insoluble nuclear substructure where transcription occurs.

Animals↗

PubMed1999

Application of exogenously regulatable promoter systems to transgenic models for the study of aging.

Transgenic mouse and gene knockout technologies offer powerful tools for dissecting the roles of specific genes in the process of aging. Tke interpretation of the results of such studies is limited, however, by the fact that the gene of interest of over- or underexpressed throughout the life span of the animal model. Among other problems, this situation makes it difficult to separate the effects that a specific gene has an embryological development from those that it may exert on the subsequent maturation and aging of the animal. It is also not possible with these methods alone to alter the expression of genes in an age-dependent fashion and to assess the effects of these alterations on the aging process. This capacity would be of particular interest in studying genes which are thought to have a role in regulating physiological homeostasis. Because they offer the opportunity to activate or render inactive the expression of genes at will, exogenously regulatable promoter systems, particularly when used in combination with traditional transgenic or gene knockout approaches, provide a new and potentially very powerful tool for studying the effect of selected genes on aging. This review discusses the merits and limitations of the application of either the tetracycline-regulatable promoter system, the RU 486-inducible promoter system, or the ecdysone-inducible promoter system to exogenously regulate the expression of a transcriptionally linked gene and to thus assess the effect of that gene on aging.

Aging↗

PubMed1998

Functional subnuclear partitioning of transcription factors.

After many years of reductionistic approaches to characterize molecular mechanisms involved in transcription, the number of factors recognized to take part in this process has increased remarkably and continues to grow. When considering posttranslational modifications in conjunction with the large number of factors involved in modulating the activity of transcription complex components, the overall intricacy becomes staggering. After two decades of intensive molecular investigations, there has been a concerted effort to integrate these findings with cellular approaches to understand transcription on a more global level. This sort of reasoning actually revisits studies of approximately 20 years ago that considered the functional consequences of steroid receptor association with nuclear structure. With an abundance of new molecular probes and increasingly powerful instruments to detect them in fixed and, more recently, live cells, the issue of functional subnuclear organization is receiving increased attention. In this report, we focus on advances in characterizing the functional significance of transcription factor association with the nucleoskeleton. In particular, we consider recent biochemical and "molecular morphology" data that point to the importance of dynamic spatial and solubility partitioning of gene regulators with nuclear architecture.

Cell Nucleus↗

PubMed1998

The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment.

The BRCA2 gene was identified based on its involvement in familial breast cancer. The analysis of its sequence predicts that the gene encodes a protein with 3,418 amino acids but provides very few clues pointing to its biological function. In an attempt to address this question, specific antibodies were prepared that identified the gene product of BRCA2 as a 390-kDa nuclear protein. Furthermore, direct binding of human RAD51 to each of the four single 30-amino acid BRC repeats located at the 5' portion of exon 11 of BRCA2 was demonstrated. Such an interaction is significant, as BRCA2 and RAD51 can be reciprocally coimmunoprecipitated by each of the individual, specific antibodies and form complexes in vivo. Inferring from the function of RAD51 in DNA repair, human pancreatic cancer cells, Capan-1, expressing truncated BRCA2 were shown to be hypersensitive to methyl methanesulfonate (MMS) treatment. Exogenous expression of wild-type BRCA2, but not BRC-deleted mutants, in Capan-1 cells confers resistance to MMS treatment. These results suggest that the interaction between the BRC repeats of BRCA2 and RAD51 is critical for cellular response to DNA damage caused by MMS.

BRCA2 Protein↗

PubMed1997

HEC binds to the seventh regulatory subunit of the 26 S proteasome and modulates the proteolysis of mitotic cyclins.

A newly identified nuclear protein rich in leucine heptad repeats called HEC is important for mitosis. To elucidate its mechanism of action, the region containing leucine heptad repeats was used to identify cellular proteins that potentially interact with HEC. Complementary DNAs encoding several proteins including MSS1, p45, Nek2, and Smc1/Smc2, known to be important for G2/M progression, were identified. The interaction between HEC and MSS1, the seventh regulatory subunit of the 26 S proteasome, was further demonstrated by in vitro GST pull-down assays. HEC is not a part of the 26 S proteasome and interacts with MSS1 only when it is dissociated from the complex during M phase. Purified MSS1 specifically hydrolyzes ATP, an activity inhibited by HEC. In addition, HEC inhibits the proteolysis of mitotic cyclin B in vitro. Consistent with this biochemical activity, ectopic expression of HEC inhibits the degradation of mitotic cyclins after telophase, resulting eventually in cell death. These results show that HEC is a negative regulator of MSS1 and suggest that it may modulate M phase progression, in part, through the regulation of proteasome-mediated degradation of cell cycle regulatory proteins.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1996

The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor.

The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1alpha (importin-alpha), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-alpha was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.

Amino Acid Sequence↗

PubMed1996

Brain POU-er.

Developmental coordination is vital in the temporally coordinated appearance of cell types within the precise spatial architecture of the vertebrate brain and this, combined with the rich interplay between the developing brain and its target organs, is a biological problem of monumental complexity. An example is the genesis and subsequent integration of the neuroendocrine hypothalamus and the pituitary. Two recent papers use the developing hypothalamo-pituitary axis in order to gather a deeper understanding of these integrative mechanisms. In addition, they show that a sub-family of homeodomain factors, the POU-domain proteins, play a critical role in coordinating the respective ontogenies of the hypothalamus and the pituitary.

Animals↗

PubMed1996

3.6 kb of the 5' flanking DNA activates the mouse tyrosine hydroxylase gene promoter without catecholaminergic-specific expression.

The tyrosine hydroxylase (TH) gene is expressed exclusively in cells and neurons that synthesize and release L-DOPA or catecholamines. To further understand the molecular genetic mechanisms that regulate this cell-type specific expression, a chimeric gene was prepared by linking 3.6 kb of the 5' flanking DNA of the mouse TH gene, including the +1 initiation site for transcription, to an E. coli beta-galactosidase reporter. This fusion gene (TH3.6LAC) was used to prepare transgenic mice, and the tissue distribution of expression of TH3.6LAC was determined by the measurement of beta-galactosidase enzymatic activity and/or by the detection of the transcription product of the chimeric gene by RNase protection assays. In two separate founder lines, TH3.6LAC expression was observed in every region of the brain that was examined, including the olfactory bulb, brainstem, cerebellum, diencephalon, hippocampus, striatum, and cerebral cortex. Expression of TH3.6LAC was observed in the adrenal gland of one founder line but not in the other. TH3.6LAC activation was undetectable in peripheral organs that were examined, including the liver, heart, salivary gland, kidney, lung, and spleen. Although 3.6 kb of the 5' regulatory DNA of the mouse TH gene is sufficient to activate the TH fusion gene in the mouse, it is not enough to restrict its expression to catecholaminergic cells.

Adrenal Glands↗

PubMed1995

Pit-1 exhibits a unique promoter spacing requirement for activation and synergism.

The developmentally regulated Pit-1 transcription factor is involved in the activation of prolactin, growth hormone, and TSH beta expression. Using templates with spacing mutations to program an in vitro transcription system, the activity of a single Pit-1 proximal binding site within the rat prolactin promoter was shown to have a unique bimodal distance requirement. Transcription activity rapidly decreased with each 5-base pair (bp) addition to the spacing between the binding site and the TATA box. When positioned 20 bp upstream from its normal -36 position in the prolactin promoter, the activity of the Pit-1 binding site is reduced to basal levels. Placement of the site at a position 30 bp upstream resulted in a return of Pit-1-mediated activation. Using transient transfection assays in GH3 cells, the prime bimodal sites are also a requirement for optimum expression of chimeric prolactin-luciferase reporter constructs. Interestingly, optimal synergism of transcription in vivo by the prolactin distal enhancer, containing four Pit-1 binding sites and an estrogen-responsive element, is also sensitive to the placement of the proximal Pit-1 binding site. These data have important implications for Pit-1 activator function in pituitary cells and for general models of transcription synergism.

Animals↗

PubMed1995

Rat Pit-1 stimulates transcription in vitro by influencing pre-initiation complex assembly.

The anterior pituitary-specific transcription factor, Pit-1, activates prolactin, growth hormone, TSH beta, growth hormone receptor genes and autoregulates the pit-1 gene. Its mechanism of transcription activation is unknown. Using immobilized DNA templates and order-of-addition transcription assays, it is shown that Pit-1 is required during pre-initiation complex assembly to activate the prolactin gene in vitro. Using prolactin promoters containing point mutations in the distal TATA box, it is also demonstrated that Pit-1 activation in vitro is not mediated simply by repressing the upstream, alternative promoter. Experiments show that a preformed class II pre-initiation complex is refractory to Pit-1 influence. The data indicate that Pit-1, and perhaps other members of the POU-protein family, activate transcription by influencing the type pre-initiation complex assembled on target promoters.

Animals↗

PubMed1993

The expression of transferrin mRNA in the salivary glands and other tissues of baboons.

In order to determine whether all the extrinsic salivary glands synthesize transferrin mRNA, the polyadenylated ribonucleic acids [poly(A)+ RNAs] from parotid, submandibular, and sublingual glands, liver, midbrain, testis, spleen, heart, kidney, and the mucosae of oesophagus and stomach from adult male baboons were analysed, using oligo(dT)-cellulose chromatography, agarose gel electrophoresis, followed by transfer of the mRNAs to nitrocellulose filters and identification with transferrin and tubulin cDNA probes. Transferrin and tubulin mRNAs were visualized by autoradiography and analysed by measuring specific activity from beta emitting nuclides following transfer to nitrocellulose filters and hybridizing with [alpha-32P]-labelled human transferrin and tubulin cDNA probes. The results indicate that transferrin mRNA is present in all the extrinsic salivary glands (submandibular, sublingual, parotid) of baboons.

Animals↗

PubMed1991

A Pit-1 binding site 3' to the transcription start site inhibits transcription elongation in vitro.

The transcription factor Pit-1 can autoregulate its own expression by interacting with binding sites associated with the Pit-1 gene. One of these binding sites is located within the transcribed region of the gene and appears to be responsible for negatively regulating Pit-1 transcription. Using unique constructions in an in vitro transcription system, we show here that binding of Pit-1 to its recognition element can impede the progress of transcription elongation through the occupied site. We propose that this is a viable mechanism for Pit-1 autoregulation.

Animals↗