PARENT SESSION

(M433) EXPRESSION AND LOCALIZATION OF BROMODOMAIN PROTEIN 4 (BRD4) DURING OOCYTE MATURATION AND FERTILIZATION IN MICE.

Nagashima, Takashi¹, Maruyama, Tetsuo¹, Uchida, Hiroshi¹, Masuda, Hirotaka¹, Masanori, Ono¹, Arase, Toru¹, Asada, Hironori¹, Ozato, Keiko², Yoshimura, Yasunori¹, ¹ Keio University School of Medicine, Tokyo, Japan² NICHD, NIH, Bethesda, MD

ABSTRACT- Bromodomains are evolutionarily conserved protein modules to bind to acetylated histones. Nuclear protein Brd4 carries two tandem bromodomains and associates with condensed chromosomes through its binding to acetylated H3 and H4 during mitosis when many other nuclear regulatory factors were released into the cytoplasm, suggesting its role in the transmission of relevant information across cell division. In addition, embryos nullizygous for Brd4 die shortly after implantation and are compromised in their ability to maintain an inner cell mass. The objective of this study was to investigate the expression and localization of Brd4 in conjunction with histone acetylation during oocyte maturation and fertilization. Oocytes were collected from superovulated immature BDF1 mice and cultured in TYH medium. The embryos were prepared by in vitro fertilization using oocytes and sperm from BDF1 mice and cultured in mW medium. Embryos at the 1-cell, 2-cell, and 4-cell stage were collected 5, 30, and 48 hr after insemination, respectively. The expression level of Brd4 protein was gradually increased after the second meiosis, as determined by immunoblotting with anti-Brd4 antibody. Immunofluorescence using anti-Brd4 antibody revealed that Brd4 was preferentially localized in the nucleus of GV oocytes, 1 cell-, 2 cell-, 4 cell-, and 8 cell-stage embryos. In contrast, Brd4 was dissociated from the condensed chromosome and dispersed into the cytoplasm of GVBD-, MI-, MII-stage oocytes when histone H3 and H4 became hypoacetylated, as determined by immunofluorecense using specific antibodies against the individual acetylated histones. Treatment of the GVBD-, MI- and MII-stage oocytes with trichostatin A (TSA), a potent and specific inhibitor of histone deacetylases, dramatically induced the hyperacetylation of their condensed chromosomes; however, Brd4 remained dissociated from the chromosomes. Our data indicate that Brd4 may behave in a different way during meiosis and that the acetylation status of the histones may not be the sole determinant of localizing Brd4 to the condensed chromosomes. Meiosis-specific modification of the chromatin structure other than histone acetylation may be required for the interaction between Brd4 and the chromatin presumably leading to the appropriate transmission of the cell memory.

KEY WORDS: bromodomain, histone, acetylation, meiosis