Xiaoting Zhang

Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p.

CAP1 encodes a basic region‐leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant‐regulated localization of Yap1p to the nucleus requires the presence of a carboxy‐terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Δyap1 S. cerevisiae strain. Whereas a Yap1p‐responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p‐expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post‐translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.

New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220

The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.

Saccharomyces cerevisiae Multidrug Resistance Gene Expression Inversely Correlates with the Status of the F0Component of the Mitochondrial ATPase

Loss of the mitochondrial genome (ρ0 cell) or elimination of the mitochondrial inner membrane protein Oxa1p causes a dramatic increase in expression of the ATP binding cassette transporter-encoding gene PDR5 in the yeast Saccharomyces cerevisiae. This increase in gene expression occurs via activation of the function of the Cys6-Zn(II)2 cluster transcription factor Pdr3p, which in turn autoregulates expression of its structural gene. Surprisingly, the acquisition of PDR5-dependent multidrug resistance occurs at a very high frequency, consistent with the appearance of ρ− cells in a fermentatively growing culture (∼2%). The degree of activation of Pdr3p target genes was found to vary considerably and to be influenced by the presence of the homologous protein, Pdr1p. Mutagenesis and overexpression studies provided evidence that the control of Pdr3p expression was the major control point of this transcription factor by mitochondrial retrograde signaling. Because both ρ0 and oxa1mutant cells have multiple defects including loss of normal respiratory chain function and oxidative phosphorylation, a series of mutant strains with more selective defects in mitochondrial function was employed to identify the molecular signal that triggersPDR5 transcriptional activation. Only mutations that influenced the functional status of the F0 subunit of the mitochondrial ATPase were found to lead to activation ofPDR5 expression.

Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand-dependent interactions of the MED1 subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is little information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice. Interestingly, these mice are both viable and fertile and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. Consistent with this phenotype, as well as loss of the strong ligand-dependent estrogen receptor (ER)α-Mediator interaction, expression of a number of known ERα-regulated genes was down-regulated in MED1-mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen-stimulated mammary duct growth in MED1-mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor- and cell-specific in vivo role for MED1 LxxLL motifs, through Mediator-ERα interactions, in mammary gland development.

Cross-talk between HER2 and MED1 Regulates Tamoxifen Resistance of Human Breast Cancer Cells

Despite the fact that most breast cancer patients have estrogen receptor (ER) α-positive tumors, up to 50% of the patients are or soon develop resistance to endocrine therapy. It is recognized that HER2 activation is one of the major mechanisms contributing to endocrine resistance. In this study, we report that the ER coactivator MED1 is a novel cross-talk point for the HER2 and ERα pathways. Tissue microarray analysis of human breast cancers revealed that MED1 expression positively correlates most strongly with HER2 status of the tumors. MED1 was highly phosphorylated, in a HER2-dependent manner, at the site known to be critical for its activation. Importantly, RNAi-mediated attenuation of MED1 sensitized HER2-overexpressing cells to tamoxifen treatment. MED1 and its phosphorylated form, but not the corepressors N-CoR and SMRT, were recruited to the ERα target gene promoter by tamoxifen in HER2-overexpressing cells. Significantly, MED1 attenuation or mutation of MED1 phosphorylation sites was sufficient to restore the promoter recruitment of N-CoR and SMRT. Notably, we found that MED1 is required for the expression of not only traditional E2-ERα target genes but also the newly described EGF-ERα target genes. Our results additionally indicated that MED1 is recruited to the HER2 gene and required for its expression. Taken together, these findings support a key role for MED1 in HER2-mediated tamoxifen resistance and suggest its potential usage as a therapeutic target to simultaneously block both ERα and HER2 pathways for the treatment of this type of endocrine resistant breast cancer.

A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism

As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet–induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1–nuclear receptor interactions.

AP-2 regulates the transcription of estrogen receptor (ER)-beta by acting through a methylation hotspot of the 0N promoter in prostate cancer cells.

We reported previously that the loss of expression of estrogen receptor (ER)-β during the development of prostate cancer (PCa) is associated with methylation of a CpG island located in the 5′-flanking sequence of the 0N promoter. Three methylation hotspots, referred to as centers 1, 2 and 3, were identified in the CpG island. In this study, we demonstrated that a 581-bp region with these three centers within it is sufficient for the promoter activity in PCa cells. Deletion analyses indicated that center 1 (16 bp), with a putative activator protein-2 (AP-2) binding site, is essential for gene transactivation. Chromatin immunoprecipitation assays showed that AP-2α occupies a short sequence containing center 1. Forced expression of AP-2α or -2γ, but not -2β, increased activity of the ERβ 0N promoter and the accumulation of mRNA. Conversely, siRNA-mediated AP-2α and -2γ knockdown reduced levels of ERβ transcript and promoter activity. Quantitative reverse transcription–PCR showed that AP-2α and -2γ are the predominant transcripts expressed in PCa cells, and levels of ERβ transcript correlate with levels of these AP-2 transcripts among different PCa cell lines. These results provide the first evidence that ERβ is an AP-2-regulated gene. They also support the hypothesis that certain cis-acting elements are methylation hotspots susceptible to epigenetic modifications during cancer progression.

Cross-talk between Transcriptional Regulators of Multidrug Resistance in Saccharomyces cerevisiae

Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys6-Zn(II)2 transcription factors Pdr1p and Pdr3p. Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance. Previous work has identified a novel Cys6-Zn(II)2 transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide. In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding geneYOR1. Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription. We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated. Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo. Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.

MiR-155 at the heart of oncogenic pathways

MicroRNAs are increasingly being recognized as oncogenes and tumor suppressors in cancer. MicroRNA-155 (miR-155) is an established oncomiR in breast cancer and regulates several pro-oncogenic pathways. In light of this, Chiang’s group has discovered a novel pathway regulated by miR-155. MiR-155 directly targets the VHL tumor suppressor and, by doing so, promotes the activity of HIF transcription factors and angiogenesis. This pathway appears to be particularly relevant in triple-negative breast cancer.