A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F.

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls.

The human hepatitis B virus (HBV) HBx protein is a small transcriptional activator that is essential for virus infection. HBx is thought to be involved in viral hepatocarcinogenesis because it promotes tumorigenesis in transgenic mice. HBx activates the RAS-RAF-mitogen-activated protein (MAP) kinase signaling cascade, through which it activates transcription factors AP-1 and NF-kappa B, and stimulates cell DNA synthesis. We show that HBx stimulates cell cycle progression, shortening the emergence of cells from quiescence (G0) and entry into S phase by at least 12 h, and accelerating transit through checkpoint controls at G0/G1 and G2/M. Compared with serum stimulation, HBx was found to strongly increase the rate and level of activation of the cyclin-dependent kinases CDK2 and CDC2, and their respective active association with cyclins E and A or cyclin B. HBx is also shown to override or greatly reduce serum dependence for cell cycle activation. Both HBx and serum were found to require activation of RAS to stimulate cell cycling, but only HBx could shorten checkpoint intervals. HBx therefore stimulates cell proliferation by activating RAS and a second unknown effector, which may be related to its reported ability to induce prolonged activation of JUN or to interact with cellular p53 protein. These data suggest a molecular mechanism by which HBx likely contributes to viral carcinogenesis. By deregulating checkpoint controls, HBx could participate in the selection of cells that are genetically unstable, some of which would accumulate unrepaired transforming mutations.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

The BCL2 major breakpoint region is a sequence- and cell-cycle-specific binding site of the Ku antigen.

The majority of translocations involving BCL2 are very narrowly targeted to three breakpoint clusters evenly spaced over a 100-bp region of the gene's terminal exon. We have recently shown that the immediate upstream boundary of this major breakpoint region (mbr) is a specific recognition site for single-strand DNA (ssDNA) binding proteins on the sense and antisense strands. The downstream flank of the mbr is a helicase binding site. In this report we demonstrate that the helicase and ssDNA binding proteins show reciprocal changes in binding activity over the cell cycle. The helicase is maximally active in G1 and early S phases; the ssDNA binding proteins are maximally active in late S and G2/M phases. An inhibitor of helicase binding appears in late S and G2/M. Finally, at least one component of the helicase binding complex is the Ku antigen. Thus, a protein with helicase activity implicated in repair of double-strand breaks, variable (diversity) joining recombination, and, potentially, cell-cycle regulation is targeted to the BCL2 mbr.

Crystal structure of the cell cycle-regulatory protein suc1 reveals a beta-hinge conformational switch.

The Schizosaccharomyces pombe cell cycle-regulatory protein suc1, named as the suppressor of cdc2 temperature-sensitive mutations, is essential for cell cycle progression. To understand suc1 structure-function relationships and to help resolve conflicting interpretations of suc1 function based on genetic studies of suc1 and its functional homologs in both lower and higher eukaryotes, we have determined the crystal structure of the beta-interchanged suc1 dimer. Each domain consists of three alpha-helices and a four-stranded beta-sheet, completed by the interchange of terminal beta-strands between the two subunits. This beta-interchanged suc1 dimer, when compared with the beta-hairpin single-domain folds of suc1, reveals a beta-hinge motif formed by the conserved amino acid sequence HVPEPH. This beta-hinge mediates the subunit conformation and assembly of suc1: closing produces the intrasubunit beta-hairpin and single-domain fold, whereas opening leads to the intersubunit beta-strand interchange and interlocked dimer assembly reported here. This conformational switch markedly changes the surface accessibility of sequence-conserved residues available for recognition of cyclin-dependent kinase, suggesting a structural mechanism for beta-hinge-mediated regulation of suc1 biological function. Thus, suc1 belongs to the family of domain-swapping proteins, consisting of intertwined and dimeric protein structures in which the dual assembly modes regulate their function.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Nuclear Domains Enriched in RNA 3′-processing Factors Associate with Coiled Bodies and Histone Genes in a Cell Cycle–dependent Manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3′-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle–dependent expression of histone genes.

Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.

The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but result from changing local cell-cell interactions. Cluster synchronization occurs before S and in the G2/M phases. Rates of cell division are neither constant nor clonal features. Cell cycle progression is linear rather than concentric. Clusters appear throughout the disc but with symmetries related to presumptive wing patterns, compartment boundaries, and vein clonal restrictions.