Molecular characterization of the tobacco SET domain protein NtSET1 unravels its role in histone methylation, chromatin binding, and segregation

Plants contain a great number of genes encoding a distinctive class of SET domain proteins which harbor a plant-specific N-terminal part together with a C-terminal part showing highest sequence similarity to the catalytic domain of the yeast CLR4, the human SUV39H1 and G9a histone-methyltransferases (HMTases). Here we show that NtSET1, a representative member of this class from tobacco, methylated both K9 and K27 of histone H3 in vitro. Ectopic expression of NtSET1, by an inducible promoter, increased the amount of dimethylated H3K9 and induced chromosome-segregation defects in tobacco BY2 cells. Deletion analyses show that the HMTase activity, the association with specific chromatin regions and with condensed chromosomes, and the cellular effects largely depended on the C-terminal region including the SET domain of the protein. Nevertheless, the N-terminal part of NtSET1 was capable of targeting the green fluorescent protein to interphase chromatin. Finally, we show that NtSET1 bound LHP1, the Arabidopsis homolog of animal heterochromatin protein 1 (HP1), and that LHP1 co-localized with heterochromatin containing high amounts of dimethylated H3K9, suggesting a role for NtSET1 in heterochromatic function. Taken together, our results provide new insights into the molecular and global chromatin-binding activities of this particular class member of plant SET domain proteins.     

Properties of a tobacco DNA methyltransferase, NtMET1 and its involvement in chromatin movement during cell division

BACKGROUND AND AIMS: Plants possess three types of DNA methyltransferase, among which methyltransferase type 1 (MET1) is considered to play a major role by maintaining the CpG methylation patterns. However, little information is available as to its enzymatic activity, interacting proteins and spatial and temporal behaviours during DNA replication. In the present study, one example, NtMET1 from tobacco plants, was selected and an analysis was made of its biochemical properties and cellular localization. METHODS: NtMET1 was expressed in Sf9 insect cells, and a purified sample was subjected to a standard in vitro methylation assay. Intramolecular interaction was examined by the yeast two-hybrid and pull-down assays. Transgenic tobacco plants (Nicotiana tabacum) over-expressing NtMET1 were constructed via Agrobacterium-mediated transformation. Cellular localization was examined by fluorescence protein fusion, which was expressed in tobacco bright yellow 2 cells. KEY RESULTS: In vitro assays showed no detectable methylation activity when both hemimethylated and unmethylated DNA samples were used as the substrate. In planta assays with over-expressing transgenic lines showed no hypermethylation but rather hypomethylation of genomc DNA. The inability of methylation was conceivably due to a tight intramolecular interaction between the N- and C-terminal regions with the catalytic domain residing on the C-terminus being completely masked. Cellular localization analyses indicated that NtMET1 localized to the nucleus in the resting stage and migrates to the cytoplasm during mitosis, particularly at metaphase. The pattern observed resembled that of Ran GTPase, and in vitro pull-down assays showed a clear interaction between NtMET1 and AtRAN3, an Arabidopsis orthologue of tobacco Ran GTPase, NtRan-A1. CONCLUSIONS: The results suggest that enzymatic activity of NtMET1 is well adjusted by its own intra/intermolecular interaction and perhaps by interactions with other proteins, one of which was found to be Ran GTPase. Results also revealed that NtMET1 becomes localized to the vicinity of chromatin with the aid of Ran GTPase during cell division, and may play an important role in progress through mitosis independently of methylation activity.     

Bestatin inhibits cell growth, cell division, and spore cell differentiation in Dictyostelium discoideum

Bestatin methyl ester (BME) is an inhibitor of Zn(2+)-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn(2+)-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA.     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.     

Inhibition of MAP2 expression affects both morphological and cell division phenotypes of neuronal differentiation

Expression of the differentiated neuronal phenotype is typically manifest in several properties: distinct morphologies and organizations of the underlying cytoskeleton; appearance of specific macromolecules; and cessation of cell division. All of these properties are induced in undifferentiated embryonal carcinoma cells exposed to retinoic acid. We show here that the mRNA and protein for the microtubule component MAP2 is also induced by retinoic acid. Stable transfectants of undifferentiated cells, constitutively expressing MAP2 antisense RNA, show significantly reduced levels of MAP2 antisense RNA, show significantly reduced levels of MAP2 protein upon induction compared with controls. These cells do express other neuronal markers, but they do not undergo normal morphological differentiation nor do they withdraw from the cell cycle. The results suggest that MAP2 expression may be necessary for both neurite extension and cessation of cell division.     

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.     

Ectopic expression of the ErbB-3 binding protein ebp1 inhibits growth and induces differentiation of human breast cancer cell lines

Ebp1, an ErbB-3 binding protein, translocates from the cytoplasm to the nucleus of human breast cancer cells after treatment with the ErbB-3 ligand, heregulin. The purpose of these studies was to examine the effects of ectopic expression of ebp1 on the biological properties of human ErbB-3-expressing breast carcinoma cell lines. Ectopic expression of ebp1 in ErbB-2, ErbB-3-expressing breast carcinoma cell lines resulted in inhibition of colony formation, a decreased proliferation rate, an accumulation of cells in the G2/M phase of the cell cycle, and suppression of growth in soft agar. Ectopic expression of ebp1 led to a more differentiated phenotype in AU565 breast cancer cells, as evidenced by increased expression of lipid droplets and of the milk protein casein. Basal phosphorylation of extracellular regulated kinases (Erks) 1 and 2, kinases activated by heregulin treatment, was also observed in ebp1 transfectants. The promoter for the intercellular adhesion molecule-1 gene, a heregulin-inducible gene, was constitutively activated in ebp1 transfectants as determined by reporter construct analysis. These data demonstrate that ectopic expression of the ErbB-3 binding protein Ebp1 inhibits proliferation and induces differentiation of ErbB-2, ErbB-3-expressing human breast carcinoma cell lines.     

A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds

Phytochrome‐interacting factor 1 (PIF1) inhibits light‐dependent seed germination. The specific function of PIF1 in seed germination is partly due to its high level of expression in imbibed seeds, but the associated regulatory factors have not been identified. Here we show that mutation of the early flowering in short days (EFS) gene, encoding an H3K4 and H3K36 methyltransferase, decreases the level of H3K36me2 and H3K36me3 but not H3K4me3 at the PIF1 locus, reduces the targeting of RNA polymerase II to the PIF1 locus, and reduces mRNA expression of PIF1 in imbibed seeds. Consistently, the efs mutant geminated even under the phyB_off condition, and had an expression profile of PIF1 target genes similar to that of the pif1 mutant. Introduction of an EFS transgene into the efs mutant restored the level of H3K36me2 and H3K36me3 at the PIF1 locus, the high‐level expression of PIF1 mRNA, the expression pattern of PIF1 target genes, and the light‐dependent germination of these seeds. Introduction of a PIF1 transgene into the efs mutant also restored the expression pattern of PIF1 target genes and light‐dependent germination in imbibed seeds, but did not restore the flowering phenotype. Taken together, our results indicate that EFS is necessary for high‐level expression of PIF1 mRNA in imbibed seeds.     

Ectopic activation of Dpp signalling in the male Drosophila germline inhibits germ cell differentiation

The mechanisms that control differentiation of stem cells to specialised cell types probably include factors intrinsic to stem cells as well as extrinsic factors produced by the microenvironment of the stem cell niche. The Drosophila male germline is renewed from a population of stem cells located in the apical tip of the adult testis. The morphological relationship between germline stem cells and their surrounding somatic cells is well understood but the factors that regulate stem cell proliferation and differentiation are still being uncovered. This study examined the effect of stimulating Dpp signalling directly in male germ cells. Ectopic Dpp or Activin signalling resulted in overproliferation of both stem cell-like and spermatogonial-like cells in the apical region of the testis. A third cell population that expressed stem cell markers was seen to proliferate in the distal testis when Dpp signalling was either stimulated or repressed in germline stem cells.     

Asymmetric cell division in land plants and algae: the driving force for differentiation

Asymmetric cell division generates two cells with different fates and has an important role in plant development. It produces distinct cell types and new organs, and maintains stem cell niches. To handle the constraints of having immobile cells, plants possess numerous unique features to obtain asymmetry, such as specific regulators of intrinsic polarity. Although several components have not yet been identified, new findings, together with knowledge from different developmental systems, now allow us to take an important step towards a mechanistic overview of asymmetric cell division in plants and algae. Strikingly, several key regulators are used for different developmental processes, and common mechanisms can be recognized.