Cell cycle programs of gene expression control morphogenetic protein localization

Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms.     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

Cyclin E expression during development in Caenorhabditis elegans

Our interest in the coordination of cell cycle control and differentiation has led us to investigate the Caenorhabditis elegans cye-1 gene encoding the G(1) cell cycle regulator cyclin E. We have studied the expression and function of cye-1 by using monoclonal antibodies directed against CYE-1 protein, cye-1::GFP reporter genes, and a cye-1 chromosomal deletion mutation. We show that a ubiquitous embryonic pattern of expression becomes restricted and dynamic during postembryonic development. Promoter analysis reveals a relatively small region of cis-acting sequences that are necessary for the complex pattern of expression of this gene. Our studies demonstrate that two other G(1) cell cycle genes, encoding cyclin D and CDK4/6, have similarly compact promoter requirements. This suggests that a relatively simple mechanism of regulation may underlie the dynamic developmental patterns of expression exhibited by these three G(1) cell cycle genes. Our analysis of a new cye-1 deletion allele confirms and extends previous studies of two point mutations in the gene.     

Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle.

Grr1 protein of the yeast Saccharomyces cerevisiae is a central component of a glucose signal transduction mechanism responsible for glucose-induced gene expression. It is required for glucose-stimulated regulation of Rgt1, a repressor of several glucose-induced HXT genes. Grr1 also plays a role in regulating the cell cycle, because it is required for degradation of the G1 cyclins Cln1 and Cln2. We discovered that Grr1 physically interacts with Skp1, a protein that has been implicated in a ubiquitin-conjugating enzyme complex that targets for degradation the cell cycle regulators Cln1 and Cln2, and the cyclin-dependent kinase inhibitor Sic1. Thus, Grr1 may regulate the cell cycle and glucose-induced gene expression via ubiquitin-mediated protein degradation. Consistent with this idea, Skp1, like Grr1, was found to be required for glucose-induced HXT gene expression. Two functional domains of Grr1 are required for its interaction with Skp1: 12 leucine-rich repeats (LRR) and an adjacent F-box. The Grr1-Skp1 interaction is enhanced by high levels of glucose. This could provide yeast with a mechanism for coupling nutrient availability to gene expression and cell cycle regulation.     

Protein kinase cascades in meiotic and mitotic cell cycle control

Eukaryotic cell cycle progression during meiosis and mitosis is extensively regulated by reversible protein phosphorylation. Many cell surface receptors for mitogens are ligand-stimulated protein-tyrosine kinases that control the activation of a network of cytoplasmic and nuclear protein-serine (threonine) kinases. Over 30 plasma membrane associated protein-tyrosine kinases are encoded by proto-oncogenes, i.e., genes that have the potential to facilitate cancer when disregulated. Proteins such as ribosomal protein S6, microtubule-associated protein-2, myelin basic protein, and casein have been used to detect intracellular protein-serine (threonine) kinases that are activated further downstream in growth factor signalling transduction cascades. Genetic analysis of yeast cell division control (cdc) mutants has revealed another 20 or so protein-serine (threonine) kinases. One of these, specified by the cdc-2 gene in Schizosaccharomyces pombe, has homologs that are stimulated during M phase in maturing sea star and frog oocytes and mammalian somatic cells. Furthermore, during meiotic maturation in these echinoderm and amphibian oocytes, this is followed by activation of many of the same protein-serine (threonine) kinases that are stimulated when quiescent mammalian somatic cells are prompted with mitogens to traverse from G0 to G1 phase. These findings imply that a similar protein kinase cascade may oversee progression at multiple points in the cell cycle.     

Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis

We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G(1)/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G(1)/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G(1)/S, and the second group, which included the mitotic activities, was regulated at G(2). The activation of the G(2) genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

Protein expression in relation to the cell cycle of exponentially growing human prostatic epithelial cells

This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short-term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate-specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post-mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2 + M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.