Global Gene Silencing is Caused by the Dissociation of RNA Polymerase II from DNA in Mouse Oocytes

Abstract

Abstract. As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 μm). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP II from the DNA. We also observed dissociation of RPB1 from the DNA in fullgrown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte

“…Germinal vesicle stage oocytes were cultured in medium containing the phosphodiesterase inhibitor milrinone to prevent germinal vesicle breakdown. Consistent with previous studies, the majority of pre-ovulatory oocytes recovered after gonadotropin stimulation exhibit a surrounded nucleolus (SN) configuration, which is associated with a more advanced stage of nuclear maturation and high developmental potential (Abe et al, 2010, De La Fuente, 2006, De La Fuente and Eppig, 2001, Miyara, 2003, Wickramasinghe et al, 1991, Zuccotti, 1995. Control SN oocytes exhibited the characteristic chromatin configuration in which heterochromatin domains are wrapped around the nucleolus and euchromatin fibers form a variable number of DNA clusters that are readily stained with histone H3 di-methylated at lysine 4, a histone modification associated with euchromatin regions (H3K4me2; Fig.…”

“…For example, fully-grown oocytes exhibit a striking large-scale chromatin remodeling event leading to the formation of a transcriptionally quiescent nuclear environment in preparation for accurate chromosome condensation and segregation during meiosis (Abe et al, 2010, De La Fuente, 2006, De La Fuente and Eppig, 2001, Miyara, 2003, Wickramasinghe et al, 1991, Zuccotti, 1995. As the growing oocyte acquires both meiotic and developmental competence, a decondensed and transcriptionally active nucleus that exhibits a characteristic non surrounded nucleolus (NSN) configuration is progressively remodeled into a condensed, transcriptionally quiescent chromatin state called the surrounded nucleolus (SN) configuration when centromeric satellite DNA forms a prominent heterochromatin rim around the nucleolus in a configuration that might be required for proper meiotic progression (Abe et al, 2010, De La Fuente, 2006, De La Fuente and Eppig, 2001, Wickramasinghe et al, 1991, Zuccotti, 1995.…”

Control of Oocyte Growth and Development by Intercellular Communication Within the Follicular Niche

“…Although the events of chromatin remodeling and transcriptional silencing are temporally correlated, they do not strictly depend on each other (Abe et al 2010;Andreu-Vieyra et al 2010;De La Fuente 2006). For example, the dissociation of RNA polymerase II from DNA, an indication of transcriptional silencing, is not dependent on the NSN-SN transition (Abe et al 2010).…”

Polycomb function during oogenesis is required for mouse embryonic development

“…We observed that transcription is properly shut down in Ring1/Rnf2 dm GV oocytes, as assessed by immunofluorescence staining for RNA polymerase II (RNAPII) (Abe et al 2010) as well as microinjected BrUTP, a ribonucleotide analog incorporated into nascent RNA (Supplemental Fig. S5A,B).…”