Biomedical subjects

Rino Cella

Publications and source records attributed to Rino Cella.

6 recordsLinked to original sources

PubMed2006

Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis.

The proliferating cell nuclear antigen (PCNA) functions as a sliding clamp for DNA polymerase, and is thus a key actor in DNA replication. It is also involved in DNA repair, maintenance of heterochromatic regions throughout replication, cell cycle regulation and programmed cell death. Identification of PCNA partners is therefore necessary for understanding these processes. Here we identify two Arabidopsis SET-domain proteins that interact with PCNA: ATXR5 and ATXR6. A truncated ATXR5Deltaex2, incapable of interacting with PCNA, also occurs in planta. ATXR6, upregulated during the S phase, is upregulated by AtE2F transcription factors, suggesting that it is required for S-phase progression. The two proteins differ in their subcellular localization: ATXR5 has a dual localization in plastids and in the nucleus, whereas ATXR6 is solely nuclear. This indicates that the two proteins may play different roles in plant cells. However, overexpression of either ATXR5 or ATXR6 causes male sterility because of the degeneration of defined cell types. Taken together, our results suggest that both proteins may play a role in the cell cycle or DNA replication, and that the activity of ATXR5 may be regulated via its subcellular localization.

Amino Acid Motifs↗

PubMed2006

Interplay between Arabidopsis activating factors E2Fb and E2Fa in cell cycle progression and development.

Eukaryotic E2Fs are conserved transcription factors playing crucial and antagonistic roles in several pathways related to cell division, DNA repair, and differentiation. In plants, these processes are strictly intermingled at the growing zone to produce postembryonic development in response to internal signals and environmental cues. Of the six AtE2F proteins found in Arabidopsis (Arabidopsis thaliana), only AtE2Fa and AtE2Fb have been clearly indicated as activators of E2F-responsive genes. AtE2Fa activity was shown to induce S phase and endoreduplication, whereas the function of AtE2Fb and the interrelationship between these two transcription factors was unclear. We have investigated the role played by the AtE2Fb gene during cell cycle and development performing in situ RNA hybridization, immunolocalization of the AtE2Fb protein in planta, and analysis of AtE2Fb promoter activity in transgenic plants. Overexpression of AtE2Fb in transgenic Arabidopsis plants led to striking modifications of the morphology of roots, cotyledons, and leaves that can be ascribed to stimulation of cell division. The accumulation of the AtE2Fb protein in these lines was paralleled by an increased expression of E2F-responsive G1/S and G2/M marker genes. These results suggest that AtE2Fa and AtE2Fb have specific expression patterns and play similar but distinct roles during cell cycle progression.

Arabidopsis↗

PubMed2003

Arabidopsis MBD proteins show different binding specificities and nuclear localization.

Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control. In plants DNA methylation affects both symmetric and asymmetric cytosines by means of different DNA-methyltransferases. In vertebrates these modifications are interpreted by a group of proteins (methylated DNA-binding domain proteins, MBDs) able to specifically bind methylated CpG. In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is still to be completed. Here we present the characterization of six different MBDs from Arabidopsis. As judged by semi-quantitative RT-PCR, their expression proved to be differentially modulated in different organs. All the corresponding polypeptides, expressed in Escherichia coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only two of them (namely MBD5, 6) were able to specifically bind methylated CpG oligonucleotides. A third protein, AtMBD11, showed a strong affinity for DNA independently from the level of methylation. Moreover we were able to differentiate MBD5 and 6, despite their high homology, for their ability to recognize methylated asymmetrical sites. The binding specificity of these three AtMBD proteins was tested not only on arbitrarily chosen probes but also on the Arabidopsis E2F recognition sequence containing a single CpG site. Protoplasts transient expression experiments of GFP-fusion proteins showed for AtMBD5 and AtMBD6 a heterochromatic localization which was affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificity and might therefore have different roles in methylation-mediated transcriptional silencing.

Amino Acid Sequence↗

PubMed2002

Two E2F sites in the Arabidopsis MCM3 promoter have different roles in cell cycle activation and meristematic expression.

The commitment to DNA replication is a key step in cell division control. The Arabidopsis MCM3 homologue forms part of the mini chromosome maintenance (MCM) complex involved in the initiation of DNA replication at the transition G(1)/S. Consistent with its role at the G(1)/S transition we show that the AtMCM3 gene is transcriptionally regulated at S phase. The 5' region of this gene contains several E2F consensus binding sites, two of which match the human consensus closely and whose roles have been studied here. The identity of the two sequences as E2F binding sites has been confirmed by electrophoretic mobility shift assay analyses. Furthermore the promoter is activated by AtE2F-a and AtDP-a factors in transient expression studies. One of the E2F binding sites is shown to be responsible for the G(2)-specific repression of the promoter in synchronized cell suspension cultures. In contrast, the second E2F binding site has a role in meristem-specific expression in planta as deletion of this site eliminates the expression of a reporter gene in root and apical meristems. Thus two highly similar E2F binding sites in the promoter of the MCM3 gene are responsible for different cell cycle regulation or developmental expression patterns depending on the cellular environment.

Arabidopsis↗

PubMed2002

The E2F family of transcription factors from Arabidopsis thaliana. Novel and conserved components of the retinoblastoma/E2F pathway in plants.

The E2F transcription factors are key components of the cyclin D/retinoblastoma/E2F pathway. Here we demonstrate that Arabidopsis thaliana contains six functional AtE2F genes that are all expressed in cell suspension culture but show different patterns of expression during cell cycle progression. According to their structural and functional features, the six AtE2Fs can be divided into two distinct groups; although the three members of the first group, AtE2Fa, AtE2Fb and AtE2Fc, possess all the conserved domains found in other plant and animal E2Fs, the remaining AtE2Fs are novel proteins, which reveal a duplication of the DNA binding domain but lack any other conserved region. Furthermore, the AtE2Fs of the first group are functional transcription factors that in association with AtDP proteins can recognize specifically an E2F cis-element and can transactivate an E2F-responsive reporter gene in plant cells. In contrast, the AtE2Fs of the second group can bind specifically the E2F site without interacting with DP partners but cannot activate gene expression and, instead, are able to inhibit E2F-dependent activation of gene expression in Arabidopsis cells. These findings suggest distinctive roles for the plant E2F proteins and point to a complex concerted regulation of E2F-dependent gene expression in plant cells.

Amino Acid Sequence↗

PubMed2002

Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development.

E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.

Base Sequence↗