RBBP4 and RBBP7 Regulate Histone Deacetylation During Oocyte Maturation in Mouse.

Abstract

RBBP4 and RBBP7 are members of the WD-40 protein family and originally found associated with retinoblastoma proteins. Both proteins are components of several different chromatin remodeling complexes that contain histone deacetylases (HDACs) and are involved in chromatin remodeling and transcription repression. Results of immunoblotting experiments revealed that Rbbp7 mRNA is recruited during maturation. In contrast, although Rbbp4 mRNA is not recruited, maternally-derived RBBP4 is unstable. Knockdown of RBBP4&7 either separately or in combination (using an siRNA and morpholino approach) during in vitro maturation resulted in a dramatic phenotype observed by time-lapse imaging. RBBP7 knockdown led to abnormal extrusion of a large polar body with membrane ruffling during anaphase. In contrast, RBBP4 knockdown resulted in the first polar body being resorbed. These oocytes also had multipolar spindles. When both RBBP4 and RBBP7 were knocked down, a large polar body was emitted and then resorbed. Knockdown of RBBP4 and RBBP7, either separately or in combination resulted in a significant increase in the incidence of abnormal spindle formation and chromosome misalignment on MII spindles, as well as an increased incidence of aneuploidy. Although the amount of HDAC1 and HDAC2 was not affected when either RBBP4 or RBBP7 were knocked down either separately or in combination, the maturation-associated decrease in acetylation of histone H4K8, H4K12, and H4K16 was inhibited, whereas there was no apparent effect on deacetylation of H4K5. This inhibitory effect likely underlies the observed improper chromosome alignment and segregation. These results implicate RBBP4 and RBBP7 in histone deacetylation during maturation and provide additional evidence that such deacetylation is required for proper chromosome segregation. This research was supported by a grant from the NIH (HD022681 to RMS).