Research ArticleRelocalization of human chromatin remodeling cofactor TIP48 in mitosis
Introduction
TIP48 and TIP49 are two closely related eukaryotic proteins that show remarkable conservation from yeast to man. They belong to the AAA+ family of ATPases (reviewed in [1], [2]) and show moderate homology to the bacterial RuvB, the ATP-dependent motor of the RuvAB complex that drives branch migration of Holliday junctions [3]. Originally identified by several unrelated approaches, they acquired a plethora of names in the literature (reviewed in [4], [5], [6], [7]). TIP49 (Rvb1p in yeast, RUVBL1, TIP49a, NMP238, Pontin52 and TAP54α) interacts with TIP48 (Rvb2p or Tih2p in yeast, RUVBL2, TIP49b, Reptin52, TAP54β) in vitro and in vivo and both genes are individually essential for viability in yeast [8], [9], Drosophila melanogaster [5] and Caenorhabditis elegans (http://www.wormbase.org/). The ability of TIP48 and TIP49 to hydrolyze ATP is essential for their biological function [6]. However, the biochemical nature and the molecular mechanisms of TIP48 and TIP49 activities remain unknown.
Both proteins were originally associated with transcription [4], [8], [10], [11] and it was subsequently discovered that a number of chromatin remodeling complexes contained TIP48 and TIP49: INO80 [12], SWR1 [13] in yeast and p400 [14] in animal cells. In addition, TIP48 and TIP49 are essential components of the histone acetylase/chromatin remodeling complex TIP60 [15], [16] or Nu4A [17] in higher organisms. Through a chromatin remodeling complex or complexes, yeast Rvb1p and Rvb2p regulate transcription of over 5% of yeast genes [6]. In addition, TIP48 and TIP49 appear to be involved in snoRNP assembly and nucleolar localization and trafficking [18], [19], [20], [21].
Two major oncogenic pathways in mammalian cells depend critically on TIP48 and TIP49, one involving c-Myc and another, β-catenin. Both proteins interact directly with c-Myc and constitute an essential cofactor for oncogenic transformation by c-Myc [22], for c-Myc and E2F1-dependent apoptosis [23] and E1A-dependent oncogenic transformation [14]. It was also shown that TIP48 and TIP49 play an essential role in the Wnt signaling pathway as they are required for β-catenin-dependent regulation of gene expression [5], [24], [25] and β-catenin-mediated neoplastic transformation [26]. It may be significant that the human TIP48 gene on chromosome 19 is very close to a gene cluster that is frequently amplified in breast cancers, while TIP49 maps to a region on chromosome 21 that is frequently rearranged in various types of leukemia [27].
A role in chromatin remodeling and histone acetylation most likely underpins many of the complex and seemingly unrelated aspects of the biological function of TIP48 and TIP49, particularly their role in the regulation of gene expression. Recently it was demonstrated that the two yeast TIP48/49 homologues, Rvb1p and Rvbp2, are essential for the catalytic activity of the Ino80 chromatin remodeling complex and specifically recruit the functionally essential Arp5p to the Ino80 complex [7]. It has been speculated that TIP48 and TIP49 (individually or as a complex) promote or remodel specific protein–protein (or/and protein–DNA) interactions.
The repertoire of TIP49 function was recently extended by observations that TIP49 associated with the mitotic spindle and the centrosomes via interactions with tubulin [28]. The findings suggested that TIP49 may play a specific role in mitosis which is distinct from its role in chromatin remodeling complexes.
In this work, we examined the subcellular distribution of the human TIP48 protein during the cell cycle and more specifically in mitosis. The expression levels of TIP48 protein were constant and did not fluctuate throughout the cell cycle. Surprisingly, its subcellular distribution underwent dramatic changes during M phase. Our experiments show that, like TIP49, TIP48 was excluded from the condensing chromosomes in early mitosis and some TIP48 associated with the centrosome and the mitotic spindle. Surprisingly, TIP48 showed distinct localization to the midzone during telophase and to the midbody during cytokinesis, which differed from that of TIP49. These results are novel and point to separate roles for TIP48 and TIP49 in mitosis. We suggest that TIP48 may function as a component of the central spindle of the mitotic apparatus and that TIP48 and TIP49 may have a role in nuclear and cellular remodeling during cell division.
Section snippets
Antibodies
To raise antibodies against the full length TIP48 and TIP49 proteins, histidine-tagged recombinant proteins were expressed in E. coli BL21 (DE3) Gold cells. The proteins were purified initially on metal affinity Talon resin, followed by MonoQ FPLC (TIP48) or Hydroxyapatite column chromatography (TIP49), and their purity was checked by SDS polyacrylamide gel electrophoresis. The recombinant proteins were used as antigens to immunize rabbits, and antibodies were affinity-purified using Actigel
Subcellular distribution and expression of TIP48 in the cell cycle
The distribution of TIP48 and TIP49 in the cells and their dynamics in the cell cycle could provide important clues about their biological role. Nuclear and cytoplasmic localizations have been reported in the literature and both TIP48 and TIP49 were found associated with the nuclear matrix [30], [31], [32]. However, the abundance and dynamics of the proteins in the cell cycle have not been investigated. To study the expression pattern of TIP48, we used affinity-purified antibodies raised in
Discussion
In this work, we report the subcellular distribution of the endogenous AAA+ protein TIP48 in HeLa cells during the cell cycle and, in particular, during mitosis. Our studies demonstrate that TIP48: (a) was found mainly, but not exclusively, in the nucleus of interphase cells and exhibited a particularly distinct localization at the nuclear periphery; (b) was redistributed throughout the cell in prometaphase and was completely excluded from the condensed chromosomes; (c) was redeployed to the
Acknowledgments
We are grateful to Professor L. Wagner from Vienna University for providing the anti-TIP49 W1B3 mouse monoclonal antibody and to Professor Jeremy Brockes from the Department of Biochemistry and Molecular Biology, UCL, for providing FITCH-conjugated phalloidin and other immunological reagents used in this study. We thank our colleague Dr. Anoup Kumar for help with fluorescent microscopy, Dr. Daniel Ciantar from the Confocal unit in the Department of Anatomy at UCL for the confocal images. We
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