Z. Dave Sharp

The Nuclear Localization Sequences of the BRCA1 Protein Interact with the Importin-α 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, hSRP1α (importin-α), 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-α 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.

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, bothin 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, γ-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.

Expression of BRC Repeats in Breast Cancer Cells Disrupts the BRCA2-Rad51 Complex and Leads to Radiation Hypersensitivity and Loss of G2/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 γ-irradiation, an inability to form Rad51 radiation-induced foci, and a failure of radiation-induced G2/M, but not G1/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.

Estrogen-receptor-α exchange and chromatin dynamics are ligand- and domain-dependent

We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor α (ERα) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERα- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in koff. ERα-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERα DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.

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. J. Cell. Biochem. 70:213–221, 1998.

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 (∼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. J. Cell. Biochem. 72:322–338, 1999.

Molecular dynamics and nuclear receptor function

The development of live cell and biochemical analysis methods has led to an increase in our understanding of the dynamic regulation of transcription. Live single cell studies using photobleaching techniques indicate that many proteins have a high nuclear mobility. Pioneering work using promoter array systems based on the lac operon or the mouse mammary tumor virus promoter enabled the study of chromatin structure, promoter occupancy and protein-chromatin interaction dynamics in relation to transcription. Chromatin immunoprecipitation (ChIP)-based assays allow an exhaustive analysis of the temporal recruitment of proteins to an endogenous promoter and provide evidence of cyclic protein-protein and protein-promoter interactions. Although reflecting different timescales, both ChIP and live cell studies indicate a highly dynamic control of transcription that until now has gone undetected and unappreciated.

Resistance to antiestrogen arzoxifene is mediated by overexpression of cyclin D1

Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)alpha-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERalpha-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERalpha transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to the new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERalpha in the presence of arzoxifene. In this altered conformation, ERalpha still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERalpha-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERalpha antagonist into an agonist. This can be explained by a stabilization of the ERalpha/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERalpha upon binding to antiestrogen are at the basis of resistance to antiestrogens.

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.

3.6 kb of the 5′ Flanking DNA Activates the Mouse Tyrosine Hydroxylase Gene Promoter Without Catecholaminergic-Specific Expression

Abstract: 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. coliβ‐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 β‐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.