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Biomedical subjects

Anne E Carpenter

Publications and source records attributed to Anne E Carpenter.

16 recordsLinked to original sources
PubMed2006

The Spemann organizer gene, Goosecoid, promotes tumor metastasis.

The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.

Aging↗
PubMed2006

In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication.

The transition from mitosis to meiosis is a defining juncture in the life cycle of sexually reproducing organisms. In yeast, the decision to enter meiosis is made before the single round of DNA replication that precedes the two meiotic divisions. We present genetic evidence of an analogous decision point in the germ line of a multicellular organism. The mouse Stra8 gene is expressed in germ cells of embryonic ovaries, where meiosis is initiated, but not in those of embryonic testes, where meiosis does not begin until after birth. Here we report that in female embryos lacking Stra8 gene function, the early, mitotic development of germ cells is normal, but these cells then fail to undergo premeiotic DNA replication, meiotic chromosome condensation, cohesion, synapsis and recombination. Combined with previous findings, these genetic data suggest that active differentiation of ovarian germ cells commences at a regulatory point upstream of premeiotic DNA replication.

Adaptor Proteins, Signal Transducing↗
PubMed2006

CellProfiler: image analysis software for identifying and quantifying cell phenotypes.

Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining).

Dose-Response Relationship, Drug↗
PubMed2006

Genetic architecture of Hsp90-dependent drug resistance.

Hsp90 potentiates the evolution of azole resistance in the model yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans via calcineurin. Here, we explored effectors downstream of calcineurin regulating this Hsp90-dependent trait. Using S. cerevisiae erg3 mutants as a model, we determined that both Crz1 and Hph1 modulate azole resistance.

Antifungal Agents↗
PubMed2006

Long-range directional movement of an interphase chromosome site.

Increasing evidence suggests functional compartmentalization of interphase nuclei. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions , association of some active genes with nuclear speckles or transcription "factories", and association of transcriptionally repressed genes with heterochromatic regions. Dynamic changes in chromosome compartmentalization imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility, described as "constrained diffusion". None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1-2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1-0.9 microm/min over distances of 1-5 microm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

Actins↗
PubMed2006

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Animals↗
PubMed2006

Microarrays of lentiviruses for gene function screens in immortalized and primary cells.

Here we describe lentivirus-infected cell microarrays for the high-throughput screening of gene function in mammalian cells. To create these arrays, we cultured mammalian cells on glass slides 'printed' with lentiviruses pseudotyped as vesicular stomatitis virus glycoprotein, which encode short hairpin RNA or cDNA. Cells that land on the printed 'features' become infected with lentivirus, creating a living array of stably transduced cell clusters within a monolayer of uninfected cells. The small size of the features of the microarrays (300 microm in diameter) allows high-density spotting of lentivirus, permitting thousands of distinct parallel infections on a single glass slide. Because lentiviruses have a wide cellular tropism, including primary cells, lentivirus-infected cell microarrays can be used as a platform for high-throughput screening in a variety of cell types.

Animals↗
PubMed2006

Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins.

We examined cell cycle-dependent changes in the proteome of human cells by systematically measuring protein dynamics in individual living cells. We used time-lapse microscopy to measure the dynamics of a random subset of 20 nuclear proteins, each tagged with yellow fluorescent protein (YFP) at its endogenous chromosomal location. We synchronized the cells in silico by aligning protein dynamics in each cell between consecutive divisions. We observed widespread (40%) cell-cycle dependence of nuclear protein levels and detected previously unknown cell cycle-dependent localization changes. This approach to dynamic proteomics can aid in discovery and accurate quantification of the extensive regulation of protein concentration and localization in individual living cells.

Bacterial Proteins↗
PubMed2005

Distinctive actions of membrane-targeted versus nuclear localized estrogen receptors in breast cancer cells.

Estrogens regulate multiple activities in breast cancer cells, including proliferation. Whereas these hormones are most commonly known to regulate gene transcription through direct interaction with estrogen receptors (ERs) and with specific DNA sequences of target genes, recent studies show that ER also activates a number of rapid signaling events that are initiated at the cell membrane. To study the membrane-initiated effects of estrogen and separate them from the activities initiated by the nuclear localized ER in human breast cancer cells, we generated MDA-MB-231 breast cancer cell lines that have stably integrated either the wild-type nuclear form of ER (WT-ER) or a modified, membrane-targeted ER (MT-ER) that lacks a nuclear localization sequence and is dually acylated with a myristoylation sequence at the N terminus and a palmitoylation sequence at the C terminus. We demonstrate that MT-ER is membrane localized in the absence of estradiol (E2), showing punctate membrane and cytoplasmic speckles after E2 exposure. In contrast to WT-ER, MT-ER was not down-regulated by E2 or by antiestrogen ICI 182,780 exposure, and MT-ER failed to regulate endogenous E2-responsive genes highly up-regulated by WT-ER. Cells expressing MT-ER showed a greater serum response element-mediated transcriptional response that was partially inhibited by antiestrogen ICI 182,780. The MT-ER and WT-ER differentially altered ERK1/2 and Akt activities and the proliferation of breast cancer cells in response to E2. Hence, this study reveals distinct actions of the MT-ER vs. the WT-ER in effecting estrogen actions in breast cancer cells.

Blotting, Western↗
PubMed2005

Cell microarrays and RNA interference chip away at gene function.

The recent development of cell microarrays offers the potential to accelerate high-throughput functional genetic studies. The widespread use of RNA interference (RNAi) has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technology, RNAi cell microarrays promise to increase the efficiency, economy and ease of genome-wide RNAi screens in metazoan cells.

Animals↗
PubMed2005

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Cells, Cultured↗
PubMed2005

Common effects of acidic activators on large-scale chromatin structure and transcription.

Large-scale chromatin decondensation has been observed after the targeting of certain acidic activators to heterochromatic chromatin domains. Acidic activators are often modular, with two or more separable transcriptional activation domains. Whether these smaller regions are sufficient for all functions of the activators has not been demonstrated. We adapted an inducible heterodimerization system to allow systematic dissection of the function of acidic activators, individual subdomains within these activators, and short acidic-hydrophobic peptide motifs within these subdomains. Here, we demonstrate that large-scale chromatin decondensation activity is a general property of acidic activators. Moreover, this activity maps to the same acidic activator subdomains and acidic-hydrophobic peptide motifs that are responsible for transcriptional activation. Two copies of a mutant peptide motif of VP16 (viral protein 16) possess large-scale chromatin decondensation activity but minimal transcriptional activity, and a synthetic acidic-hydrophobic peptide motif had large-scale chromatin decondensation activity comparable to the strongest full-length acidic activator but no transcriptional activity. Therefore, the general property of large-scale chromatin decondensation shared by most acidic activators is not simply a direct result of transcription per se but is most likely the result of the concerted action of coactivator proteins recruited by the activators' short acidic-hydrophobic peptide motifs.

Amino Acid Motifs↗
PubMed2004

RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells.

RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.

Animals↗
PubMed2004

Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity.

BACKGROUND: Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. METHODS: We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. RESULTS: The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this approach, we identified two subdomains within the E domain of estrogen receptor alpha capable of inducing large-scale chromatin decondensation. CONCLUSIONS: This work confirms that, like BRCA1, the activation function 2 region of the estrogen receptor has more than one distinct chromatin unfolding domain. In addition, we demonstrate the feasibility of using automated microscopy as a high-throughput screen for identifying modulators of large-scale chromatin folding. The Supplementary Material referred to in this article can be found at the CYTO Part A website (http://www.interscience.wiley.com/jpages/0196-4763/suppmat/v58A.html)

Animals↗