The Arabidopsis genome encodes structurally and functionally diverse HMGB-type proteins.

Department of Life Sciences, Aalborg University, Sohn-gaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Journal of Molecular Biology (Impact Factor: 3.96). 06/2006; 358(3):654-64. DOI: 10.1016/j.jmb.2006.02.068
Source: PubMed

ABSTRACT The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription and recombination. In addition to the previously identified HMGB1-HMGB6 proteins, the Arabidopsis genome encodes at least two other candidate family members (encoded by the loci At2g34450 and At5g23405) having the typical overall structure of a central domain displaying sequence similarity to HMG-box DNA binding domains, which is flanked by basic N-terminal and acidic C-terminal regions. Subcellular localisation experiments demonstrate that the At2g34450 protein is a nuclear protein, whereas the At5g23405 protein is found mainly in the cytoplasm. In line with this finding, At5g23405 displays specific interaction with the nuclear export receptor AtXPO1a. According to CD measurements, the HMG-box domains of both proteins have an alpha-helical structure. The HMG-box domain of At2g34450 interacts with linear DNA and binds structure-specifically to DNA minicircles, whereas the HMG-box domain of At5g23405 does not interact with DNA at all. In ligation experiments with short DNA fragments, the At2g34450 HMG-box domain can facilitate the formation of linear oligomers, but it does not promote the formation of DNA minicircles. Therefore, the At2g34450 protein shares several features with HMGB proteins, whereas the At5g23405 protein has different characteristics. Despite the presence of a region with similarity to the nucleosome-binding domain typical of HMGN proteins, At2g34450 does not bind nucleosome particles. In summary, our data demonstrate (i) that plant HMGB-type proteins are functionally variable and (ii) that it is difficult to predict HMG-box function solely based on sequence similarity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear matrix attachment regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase IIbinding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two tobacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription.
    Moleculer Cells 07/2013; 36(2). DOI:10.1007/s10059-013-0092-z · 2.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.
    PLoS ONE 03/2013; 8(3):e60644. DOI:10.1371/journal.pone.0060644 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In flowering plants, 2 sperm cells (SCs) are generated from a generative cell (GC) in the developing pollen grain or growing pollen tube, and then delivered to embryo sac to initiate double fertilization. SC development and function specialization involve the strict control of the protein (gene) expression program and coordination of diverse cellular processes. However, because methods for collecting a large amount of highly purified GCs and SCs for proteomic and transcriptomic studies from a plant are not available, molecular information about the program and the interconnections is lacking. Here, we describe a method for obtaining a large quantity of highly purified GCs and SCs from just germinated lily pollen grains and growing pollen tubes for proteomic analysis. Our observation showed that SCs had less condensed chromatin and more vacuole-like structures than GCs, and that mature SCs were arrested at the G2 phase. Comparison of SC and GC proteomes revealed 101 proteins differentially expressed in the 2 proteomes. These proteins are involved in diverse cellular and metabolic processes with preferential involvement in metabolism, the cell cycle, signaling, ubiquitin/proteasome pathway and chromatin remodeling. Impressively, almost all proteins in SCF complex-mediated proteolysis and the cell cycle were up-regulated in SCs, whereas those in chromatin remodeling and stress response were down-regulated. Our data also reveal the coordination of SCF complex mediated proteolysis, cell cycle progression and DNA repair in SC development and function specialization. This study revealed for the first time difference in protein profiles between GCs and SCs.
    Journal of Proteome Research 07/2013; 12(11). DOI:10.1021/pr400291p · 5.00 Impact Factor