Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism

A DNA Polymerase α Accessory Protein, Mcl1, Is Required for Propagation of Centromere Structures in Fission Yeast

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-ACnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase α (Polα) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-ACnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7+, which encodes a catalytic subunit of Polα. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Polα. These results suggest that Mcl1 and Polα are required for propagation of centromere chromatin structures during DNA replication

Anti-oxidative enzyme catalase controls proliferation of HaCaT cells through the regulation of heparan sulfate 2-O-sulfotransferase

Heparan sulfate (HS) chains can interact with a variety of growth factors possessing heparin-binding domains, such as fibroblast growth factors (FGFs), enabling them to transfer to their high-affinity signaling receptors on the cell surface. In this study, we demonstrated the correlation between the expression of anti-oxidative enzymes and the HS chain in the human HaCaT keratinocyte cell line. The overexpression of catalase induced an increase of anti-HS antibody (10E4) epitope expression in these cells; however the MnSOD transfectant expressed it at the same level as the control cells. Western blotting showed that the smeared bands of HSPG were obviously shifted to higher-molecular-weight in the catalase transfectants owing to glycosylation. The levels of glycosyltransferases (XT-II, EXTL2, GLCE, HS2ST1 and HS6ST1) transcripts were significantly increased in the transfectants. In contrast, siRNA-mediated repression of catalase decreased 10E4 epitope expression, the transcript level of HS2ST1, and proliferation of HaCaT cells; however it did not significantly down-regulate the transcript level of HS6ST1. In addition, siRNA-mediated repression of HS2ST1 decreased HaCaT cell proliferation, although repression of HS6ST1 did not reduce it. These findings suggest that catalase may be able to modify the HS chains in the keratinocytes through the regulation of HS2ST1 to control HaCaT cell proliferation.37th Annual ESDR Meeting 200

Impacts of a Midlatitude Oceanic Frontal Zone for the Baroclinic Annular Mode in the Southern Hemisphere

As a major mode of annular variability in the Southern Hemisphere, the baroclinic annular mode (BAM) represents the pulsing of extratropical eddy activity. Focusing mainly on subweekly disturbances, this study assesses the impacts of a midlatitude oceanic frontal zone on the BAM and its dynamics through a set of "aquaplanet" atmospheric general circulation model experiments with zonally uniform sea surface temperature (SST) profiles prescribed. Though idealized, one experiment with realistic frontal SST gradient reasonably well reproduces observed BAM-associated anomalies as a manifestation of a typical life cycle of migratory baroclinic disturbances. Qualitatively, these BAM features are also simulated in the other experiment where the frontal SST gradient is removed. However, the BAM-associated variability weakens markedly and shifts equatorward, in association with the corresponding modifications in the climatological-mean storm track activity. The midlatitude oceanic frontal zone amplifies and anchors the BAM variability by restoring near-surface baroclinicity through anomalous sensible heat supply from the ocean and moisture supply to cyclones, although the BAM is essentially a manifestation of atmospheric internal dynamics. Those experiments and observations further indicate that the BAM modulates momentum flux associated with transient disturbances to induce a modest but robust meridional shift of the polar-front jet, suggesting that the BAM can help maintain the southern annular mode. They also indicate that the quasi-periodic behavior of the BAM is likely to reflect internal dynamics in which atmospheric disturbances on both subweekly and longer time scales are involved

Hydrogen peroxide as a potential mediator of the transcriptional regulation of heparan sulphate biosynthesis in keratinocytes.

Ionizing radiation is one of the types of oxidative stress that has a number of damaging effects on cutaneous tissues. One of the histological features of radiation-induced cutaneous fibrosis is the accumulation of extracellular matrix (ECM) components, including heparan sulfate proteoglycan (HSPG), which are required for the repair of tissue damage, and operate by interacting with a variety of growth factors. In this study, we established a model of human HaCaT keratinocytes overexpressing anti-oxidative enzyme genes to elucidate the mechanism of oxidative stress leading to the accumulation of HSPG and the role of its accumulation. Catalase overexpression induced an increase in anti-HS antibody (10E4) epitope expression in these cells. Western blotting showed that the smeared bands of HSPG were obviously shifted to a higher molecular weight in the catalase transfectants due to glycosylation. After heparitinase I treatment, the core proteins of HSPG were expressed in the catalase transfectants to almost the same extent as in the control cells. In addition, the transcript levels of all the enzymes required for the synthesis of the heparan sulfate chain were estimated in the catalase transfectant clones. The levels of five enzyme transcripts - xylosyltransferase-II (XT-II), EXTL2, D-glucuronyl C5-epimerase (GLCE), HS2-O-sulfotransferase (HS2ST), and HS6-O-sulfotransferase (HS6ST) - were significantly increased in the transfectants. Moreover, hydrogen peroxide was found to down-regulate the levels of these enzymes. By contrast, siRNA-mediated repression of catalase decreased 10E4 epitope expression, the transcript level of HS2ST1, and the growth rate of HaCaT cells. These findings suggested that peroxide-mediated transcriptional regulation of HS metabolism-related genes modified the HS chains in the HaCaT keratinocytes

Cells overexpressing catalase are more sensitive to TNF : Role of superoxide anion in TNF -induced apoptosis

96th Annual Meetin