Crosstalk between the Unfolded Protein Response and Pathways That Regulate Pathogenic Development in Ustilago maydis

The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy.     

Rbf1-independent termination of E2f1-target gene expression during early Drosophila embryogenesis

The initiation and maintenance of G1 cell cycle arrest is a key feature of animal development. In the Drosophila ectoderm, G1 arrest first appears during the seventeenth embryonic cell cycle. The initiation of G1(17) arrest requires the developmentally-induced expression of Dacapo, a p27-like Cyclin E-Cdk2 inhibitor. The maintenance of G1(17) arrest requires Rbf1-dependent repression of E2f1-regulated replication factor genes, which are expressed continuously during cycles 1-16 when S phase immediately follows mitosis. The mechanisms that trigger Rbf1 repressor function and mediate G1(17) maintenance are unknown. Here we show that the initial downregulation of expression of the E2f1-target gene RnrS, which occurs during cycles 15 and 16 prior to entry into G1(17), does not require Rbf1 or p27(Dap). This suggests a mechanism for Rbf1-independent control of E2f1 during early development. We show that E2f1 protein is destroyed in a cell cycle-dependent manner during S phase of cycles 15 and 16. E2f1 is destroyed during early S phase, and requires ongoing DNA replication. E2f1 protein reaccumulates in epidermal cells arrested in G1(17), and in these cells the induction of p27(Dap) activates Rbf1 to repress E2f1-target genes to maintain a stable G1 arrest.     

Implication of mitogen-activated protein kinase in the induction of G1 cell cycle arrest and gadd45 expression by the carotenoid fucoxanthin in human cancer cells

Background

The precise mechanism of the anti-tumor action of fucoxanthin has yet to be elucidated. We previously reported that gadd45a and gadd45b might play a role in the G1 arrest induced by fucoxanthin. In the present study, we show that several MAPKs modulate the induction of gadd45 and G1 arrest.

Methods

HepG2 and DU145 cells were used. The cell cycle was analyzed using flow cytometry. Expression of gadd45 was assayed by Northern blot and/or quantitative RT-PCR analyses. Activation of MAPK was assayed by Western blot analysis.

Results

Inhibition of p38 MAPK enhanced the induction of gadd45a expression and G1 arrest by fucoxanthin in HepG2 cells. Inhibition of ERK enhanced gadd45b expression but had no effect on the induction of G1 arrest by fucoxanthin in HepG2 cells. Inhibition of SAPK/JNK suppressed the induction of gadd45a expression and G1 arrest by fucoxanthin in DU145 cells.These data suggest that gadd45a is closely related with the G1 arrest induced by fucoxanthin, and that the pattern of MAPK involvement in the induction of gadd45a and G1 arrest by fucoxanthin differs depending on the cell type.

General significance

The implication of GADD45 and MAPK involvement in the anti-tumor action of carotenoids is first described.     

Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis

In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G₂ phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.     

Mating-Type Regulation of Methyl Methanesulfonate Sensitivity in SACCHAROMYCES CEREVISIAE

Heterozygosity at the mating-type locus (MAT) in Saccharomyces cerevisiae has been shown previously to enhance X-ray survival in diploid cells. We now show that a/α diploids are also more resistant to the radiomimetic agent methyl methanesulfonate (MMS) than are diploids that are homozygous at MAT (i.e., either a/a or α/α). Log-phase a/α cultures exhibit biphasic MMS survival curves, in which the more resistant fraction consists of budded cells (those cells in the S and G2 phases of the cell cycle). Survival curves for log-phase cultures of a/a or α/α diploids have little if any biphasic nature, suggesting that the enhanced S- and G2-phase repair capacity of a/α cells may be associated with heterozygosity at MAT. The survival of cells arrested at the beginning of the S phase with hydroxyurea indicates that MAT-dependent MMS repair is limited to S and G2, whereas MAT-independent repair can occur in G1.     

Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cip1/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cip1/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.     

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G₀/G₁ while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G₀/G₁ arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.     

Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline derivatives, synthesis and biological activity

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.     

Functional Interactions between the erupted/tsg101 Growth Suppressor Gene and the DaPKC and rbf1 Genes in Drosophila Imaginal Disc Tumors

Background

The Drosophila gene erupted (ept) encodes the fly homolog of human Tumor Susceptibility Gene-101 (TSG101), which functions as part of the conserved ESCRT-1 complex to facilitate the movement of cargoes through the endolysosomal pathway. Loss of ept or other genes that encode components of the endocytic machinery (e.g. synatxin7/avalanche, rab5, and vps25) produces disorganized overgrowth of imaginal disc tissue. Excess cell division is postulated to be a primary cause of these ‘neoplastic’ phenotypes, but the autonomous effect of these mutations on cell cycle control has not been examined.

Principal Findings

Here we show that disc cells lacking ept function display an altered cell cycle profile indicative of deregulated progression through the G1-to-S phase transition and express reduced levels of the tumor suppressor ortholog and G1/S inhibitor Rbf1. Genetic reductions of the Drosophila aPKC kinase (DaPKC), which has been shown to promote tumor growth in other fly tumor models, prevent both the ept neoplastic phenotype and the reduction in Rbf1 levels that otherwise occurs in clones of ept mutant cells; this effect is coincident with changes in localization of Notch and Crumbs, two proteins whose sorting is altered in ept mutant cells. The effect on Rbf1 can also be blocked by removal of the γ-secretase component presenilin, suggesting that cleavage of a γ-secretase target influences Rbf1 levels in ept mutant cells. Expression of exogenous rbf1 completely ablates ept mutant eye tissues but only mildly affects the development of discs composed of cells with wild type ept.

Conclusions

Together, these data show that loss of ept alters nuclear cell cycle control in developing imaginal discs and identify the DaPKC, presenilin, and rbf1 genes as modifiers of molecular and cellular phenotypes that result from loss of ept.