ABSTRACT
Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Delta yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (gamma-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced gamma-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.
Subject(s)
Angiopoietins/physiology , Cell Nucleus/metabolism , DNA Repair/physiology , ATPases Associated with Diverse Cellular Activities , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Angiopoietin-Like Protein 6 , Angiopoietin-like Proteins , Ataxia Telangiectasia Mutated Proteins , Bleomycin/pharmacology , Cell Cycle Proteins/metabolism , Cell Line , Chromatin/drug effects , Chromatin/metabolism , Cytoplasm/metabolism , DNA Breaks, Double-Stranded/drug effects , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Fatty Acids, Unsaturated/pharmacology , HeLa Cells , Histones/metabolism , Humans , Karyopherins/antagonists & inhibitors , Nuclear Localization Signals/genetics , Phosphorylation/drug effects , Protein Binding , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Deletion , Transformation, Genetic , Tumor Suppressor Proteins/metabolism , Exportin 1 ProteinABSTRACT
The actin family consists of conventional actin and various actin-related proteins (Arps). Some of these Arps are localized in the nucleus, and a fraction of each of these nuclear Arps is functionally involved in chromatin remodeling and histone acetyltransferase complexes. On the other hand, in mitotic cells, the localization and function of the nuclear Arps are largely unknown. Human Arp8 (hArp8), an ortholog of yeast nuclear Arp8, was recently found to be associated with the hINO80-chromatin remodeling complex along with hArp5. Here we report that hArp8, but not hArp5, accumulates on mitotic chromosomes. This is the first example where a member of the actin family is found to be associated with mitotic chromosomes. Expression of truncated hArp8 proteins and depletion of endogenous hArp8 by RNA interference caused misalignment of mitotic chromosomes, suggesting that chromosome-associated hArp8 has a role in chromosome behavior. In contrast, depletion of hIno80 and hArp5 did not cause misalignment of chromosomes, suggesting that the role of hArp8 at mitotic chromosomes is independent of the activity of hINO80 complexes. These findings provide the first insight into a novel function of actin family members in mitosis.
