Functional analysis of the SIN3-Histone deacetylase RPD3-RbAp48-histone H4connection in the Xenopus oocyte

We investigated the protein associations and enzymatic requirements for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcription al repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuc lear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nucle ar. We cloned Xenopus RbAp48 and SIN3 and show that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histon e deacetylase activity and transcriptional repression of the TR beta A prom oter. This repression requires deacetylase activity and nuclear import of R PD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous R PD3 is not incorporated into previously described oocyte deacetylase and AT Pase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. We show that RPD3 associates,vith RbAp48 through N- and C-terminal contacts and that RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 m ay be targeted to histones through interaction with endogenous RbAp48 to di rect transcriptional repression of the Xenopus TR beta A promoter in the oo cyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors.