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Matrin3 is a highly conserved inner nuclear matrix protein involved in multiple stages of RNA metabolism. Although Matrin3 may also play a role in DNA repair, its precise roles have remained unclear. In this study, we showed that the depletion of Matrin3 led to decreased homologous recombination (HR) efficiency and increased radiation sensitivity of cells. Matrin3-depleted cells showed impaired DNA damage-dependent focus formation of RAD51, a key protein in HR. These findings suggest that Matrin3 promotes HR by regulating RAD51.

RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.

Bach1 functions as a transcriptional repressor of heme oxygenase-1 (HO-1) and the beta-globin genes. The enhancer regions of these genes contain multiple Maf recognition elements (MAREs) to which Bach1 can bind. Previous studies have shown that increased levels of heme and cadmium induce the nuclear export of Bach1, resulting in cytoplasmic accumulation. By means of a yeast two hybrid screening using Bach1 as bait, we identified the intracellular hyaluronic acid binding protein (IHABP) as a potential regulator of Bach1. IHABP is a microtubule-associated protein that may regulate the organization of the cytoskeletal network. A series of domain analyses revealed that a region of Bach1 previously implicated in cytoplasmic accumulation was necessary for IHABP-binding. A C-terminal region of IHABP was necessary for Bach1-binding. Overexpressed Bach1 colocalized with IHABP in the cytoplasm, forming fiber-like structures on microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed a dynamic nature of the Bach1-IHABP interaction in living cells. The repression of HO-1 reporter activity by Bach1 was attenuated by co-transfecting IHABP in a dose-dependent manner. Moreover, the overexpression of IHABP induced the endogenous HO-1 gene in NIH3T3 cells. The overall results suggest that IHABP regulates the subcelluar localization of Bach1 in order to fine-tune transactivation of Bach1 target genes such as HO-1.

Bach2 is a member of the BTB-basic region leucine zipper factor family and represses transcription activity directed by the TPA response element, the Maf recognition element (MARE) and the antioxidant-responsive element. Recently, it was reported that upon oxidative stress Bach2 forms nuclear foci surrounding the promyelocytic leukaemia (PML) bodies and specifically represses the transcription around the PML bodies. Here we report that expression of the silencing mediator of retinoid and thyroid receptor (SMRT) and histone deacetylase4 (HDAC4) enhances the formation of the Bach2 foci in the nuclear matrix. SMRT mediates the HDAC4 binding to Bach2, and HDAC4 facilitates the retention of Bach2 in the foci. Scratch transcription labelling and 3D-reconstruction from the confocal images demonstrated that transcription is suppressed in and around the Bach2 foci. Indeed, Bach2 bound MARE and repressed the expression from the chromosomally integrated MARE-driven reporter gene when co-expressed with SMRT and HDAC4. Our observations suggest that both SMRT and HDAC4 play an important role in nuclear retention and the Bach2 focus formation in the mammalian cell nucleus, which may contribute to the local transcription repression.