The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.The aspergilli are a ubiquitous group of filamentous fungi spanning over 200 million years of evolution. Among the over 185 aspergilli are several that have an impact on human health and society, including 20 human pathogens as well as beneficial species used to produce foodstuffs and industrial enzymes 1 . Within this genus, A. nidulans has a central role as a model organism. In contrast to most aspergilli, A. nidulans possesses a well-characterized sexual cycle and thus a well-developed genetics system. Half a century of A. nidulans research has advanced the study of eukaryotic cellular physiology, contributing to our understanding of metabolic regulation, development, cell cycle control, chromatin structure, cytoskeletal function, DNA repair, pH control, morphogenesis, mitochondrial DNA structure and human genetic diseases.We present here the genome sequence for A. nidulans, and a comparative genomics study with two related aspergilli: A. fumigatus 2 and A. oryzae 3 . A. fumigatus is a life-threatening human pathogen, and ARTICLES

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase C⑀ (PKC⑀), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKC⑀ activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKC⑀ or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKC⑀-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKC⑀-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.Chemotherapeutic failure frequently contributes to morbidity in patients diagnosed with solid tumors, such as breast cancers (1-3). Recent studies indicate that oncogenic signaling and tumor cell-specific function are directly involved in chemotherapeutic drug resistance and breast tumor progression (4 -6). A number of studies have aimed at identifying those molecules that are specifically expressed by epithelial tumor cells and correlate with metastatic behavior and chemoresistance. Among such molecules is hyaluronan (HA), 2 a major component in the extracellular matrix of most mammalian tissues (7,8). HA is a nonsulfated, unbranched glycosaminoglycan, consisting of repeating disaccharide units, D-glucuronic acid, and N-acetyl-D-glucosamine (9, 10). The biosynthesis of HA is regulated by three mammalian HA synthase isozymes, HA synthase 1, 2, and 3 (11-14). Abnormal production of HA directly contributes to aberrant cellular processes, such as transformation and metastasis (15). Furthermore, HA is digested into a variety of smaller-sized molecules by various hyaluronidases (16). Activation of extracellular matrix-degrading enzymes, such as the hyaluronidases, appears to be closely associated w...

MicroRNAs are often associated with the pathogenesis of many cancers including Head and Neck Squamous Cell Carcinoma (HNSCC). In particular, microRNA-21 (miR-21) appears to play a critical role in tumor cell survival, chemoresistance and HNSCC progression. In this study we investigated matrix hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with the stem cell markers, Nanog and Stat-3, in HNSCC cells (HSC-3 cells). Our results indicate that HA binding to CD44 promotes Nanog-Stat-3 (also tyrosine phosphorylated Stat-3) complex formation, nuclear translocation and transcriptional activation. Further analyses reveal that miR-21 is controlled by an upstream promoter containing Stat-3 binding site(s), while chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-21 expression by HA/CD44 signaling is Nanog/Stat-3-dependent in HNSCC cells. This process results in a decrease of a tumor suppressor protein (PDCD4), and an upregulation of inhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in HSC-3 cells. Treatment of HSC-3 cells with Nanog- and/or Stat-3-specific small interfering RNAs (siRNAs) effectively blocks HA-mediated Nanog-Stat-3 signaling events, abrogates miR-21 production and increases PDCD4 expression. Subsequently, this Nanog-Stat-3 signaling inhibition causes downregulation of survival protein (IAP) expression and enhancement of chemosensitivity. To further evaluate the role of miR-21 in tumor cell-specific functions, HSC-3 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and block its target functions. Our results demonstrate that anti-miR-21 inhibitor not only upregulates PDCD4 expression, but also decreases IAP expression and enhances chemosensitivity in HA-treated HNSCC cells. Together, these findings indicate that the HA-induced CD44 interaction with Nanog and Stat-3 plays a pivotal role in miR-21 production leading to PDCD4 reduction, IAP upregulation and chemoresistance in HNSCC cells. This novel Nanog/Stat-3 signaling pathway-specific mechanism involved in miR-21 production is significant for the formation of future intervention strategies in the treatment of HA/CD44-activated HNSCC.

Dysregulation of microRNAs is observed in many cancersCD44 denotes a family of cell surface glycoprotein receptors that are expressed in a variety of human solid neoplasms, particularly those classified as breast cancer (1-16). Nucleotide sequence analyses reveal that many CD44 isoforms (derived by alternative splicing mechanisms) are variants of the standard form, CD44s (17). The presence of high levels of CD44 variant (CD44v) isoforms is emerging as an important metastatic tumor marker in a number of cancers, including human breast cancers (1-16). Recent studies have shown that CD44 is also expressed in tumor stem cells that have the unique ability to initiate tumor cell-specific properties (18). In fact, CD44 appears to be an important surface marker for cancer stem cells (18). Hyaluronan (HA) 2 (a major component in the extracellular matrix of most mammalian tissues) is also rich in stem cell niches (19). All CD44 isoforms contain an HA-binding site in their extracellular domain and thereby serve as a major cell surface receptor for HA (1-16). Importantly, both CD44 and HA are overexpressed/elevated at sites of tumor attachment (1-16). HA binding to CD44 not only affects cell adhesion to the matrix components but also is involved in the stimulation of a variety of tumor cell-specific functions leading to breast cancer progression (1-16).The Src family kinases are classified as oncogenic proteins due to their ability to activate cell proliferation, spreading, and migration in many cell types, including epithelial tumor cells (20). It has been observed that the interaction between Src kinase and membrane-linked molecules regulates receptor signaling and various cellular functions (21). In fact, CD44-mediated cellular signaling has been determined to involve Src kinase family members (2,15,22). Moreover, the cytoplasmic domain of CD44 binds to c-Src kinase at a single site with high affinity (15). Most importantly, HA interaction with CD44 stimulates c-Src kinase activity, which, in turn, increases tyrosine phosphorylation of the cytoskeletal protein, cortactin. Subsequently, tyrosine phosphorylation of cortactin attenuates its ability to cross-link filamentous actin in vitro (15

The evolutionarily conserved fungal arginine attenuator peptide (AAP), as a nascent peptide, stalls the translating ribosome in response to the presence of a high concentration of the amino acid arginine. Here we examine whether the AAP maintains regulatory function in fungal, plant, and animal cell-free translation systems when placed as a domain near the N terminus or internally within a large polypeptide. Pulse-chase analyses of the radiolabeled polypeptides synthesized in these systems indicated that wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts introduced into the fungal system revealed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide-sensing features are conserved in fungal, plant, and animal ribosomes. These data provide precedents for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression.