Oocyte maturation is a complex process that prepares the oocyte to undergo dramatic changes rapidly upon fertilization (Greenstein, 2005
; Masui and Clarke, 1979
). Many hallmarks of maturation have been described for years, including nuclear envelope breakdown and cytoskeletal rearrangements, but the molecular and regulatory events associated with maturation are not well understood. Here we report that C. elegans
oocytes shut down transcription independently of signals that stimulate maturation, as indicated by Ser 5 and Ser 2 phosphorylation being undetectable in diakinetic fog-2
oocytes in the absence of sperm (; data not shown). Surprisingly, our findings also indicate that maturation signals induce the occurrence of early transcription steps in these transcriptionally “silent” cells ().
Induction of transcription steps by oocyte maturation
Firstly, we found that diakinetic oocytes accumulate Ser 5-phosphorylated Pol II in
response to maturation signals after RNAi knockdown of the CTD phosphatase FCP-1 (). Importantly, the levels of this PSer5 staining characteristically increase as fcp-1(RNAi)
oocytes develop and move distally-to-proximally (, Supplementary Table 1
), indicating that the bulk of this Ser 5 phosphorylation does not perdure from earlier stages, and is generated de novo in these diakinetic oocytes. This PSer5 staining depends upon multiple independent transcription initiation factors that associate with Pol II specifically at promoters (), suggesting that this CTD phosphorylation is generated in the context of transcription initiation steps. The intensity of this PSer5 staining is comparable to or higher than that seen in transcriptionally active diplotene oocytes (), indicating that a substantial level of this CTD phosphorylation occurs. Because this phosphorylation accumulates to high levels in maturing oocytes when the CTD phosphatase FCP-1 is lacking, we believe that this phosphorylation normally occurs during maturation but is rapidly turned over through dephosphorylation. In general, the transcription steps in which this PSer5 staining is generated do not appear to progress to the elongation phase, however, based upon the far lower levels of Ser 2 phosphorylation detected in these diakinetic oocytes ().
We also detected Ser 5 phosphorylation that was associated with transcription and maturation when we inhibited ubc-2
-dependent ubiquitylation (, ). It will be difficult to identify the molecular target of this ubiquitylation pathway, particularly because numerous transcription factors undergo ubiquitylation (Muratani and Tansey, 2003
; Somesh et al., 2005
), but these results independently corroborate the occurrence of maturation-dependent transcription steps during diakinesis, as detected in fcp-1(RNAi)
oocytes. It is intriguing that inhibition of this ubiquitylation mechanism did not result in a dramatic progressive increase in PSer5 levels as seen in fcp-1(RNAi)
oocytes (). Perhaps the ubiquitin/proteasome pathway we have identified acts at an earlier initiation step, either to limit the rate of CTD Ser 5 phosphorylation or promote Pol II recycling.
Finally, we detected robust Ser 5 but not Ser 2 phosphorylation in diakinetic oocytes in vab-1(dx31); fog-2(q71); ceh-18(mg57) and wee-1(RNAi) animals (), which undergo maturation prematurely. These last experiments are significant because they allowed us to detect this CTD phosphorylation without manipulating regulatory mechanisms that directly influence transcription. Presumably, the presence of these maturation signals at inappropriate intensity or duration during late oogenesis stages resulted in accumulation of transcription-dependent PSer5 even in the presence of the FCP-1 phosphatase.
An important implication of our findings is that FCP-1 may function as a major Ser 5 phosphatase in vivo, in addition to its previously described functions in Ser 2 dephosphorylation (Cho et al., 2001
; Meinhart et al., 2005
). When FCP-1 was lacking, transcription-associated PSer5 that was generated in response to maturation accumulated throughout the nucleus, suggesting that this modified Pol II had dissociated from the DNA. Interestingly, after fcp-1
RNAi we also observed nucleoplasmic PSer5 staining in embryonic germ cell precursors (), in which PIE-1 has been proposed to repress transcription by acting at a post-initiation step (Batchelder et al., 1999
; Zhang et al., 2003
). We speculate that an important function of FCP-1 may be to recycle Ser 5-phosphorylated Pol II that is produced during stalled or abortive transcription events, although we do not know whether in this context these events proceed to the point of generating incomplete mRNA transcripts. Importantly, fcp-1
does not seem to be required for Ser 5 phosphorylation levels to drop dramatically upon diakinesis entry in oocytes that do not proceed to maturation (fog-2(q71); fcp-1(RNAi)
oocytes; ). This suggests that in C. elegans
, oocyte transcription shutdown may not require fcp-1
and therefore does not seem to involve a wave of FCP-1-mediated CTD dephosphorylation, as may occur in Xenopus
(Palancade et al., 2001
The evidence that maturation signals induce high levels of transcription-associated CTD phosphorylation suggests that in these transcriptionally silent oocytes, many genes are accessible to and are bound by the transcription apparatus. We propose that beginning at diakinesis C. elegans
oocytes initiate and maintain transcriptional silence largely through regulation of the transcription machinery, as opposed to rendering genes globally inaccessible through chromatin changes. Accordingly, prior to ZGA embryos display histone modification markers that are associated with active chromatin (Bean et al., 2004
; Schaner et al., 2003
As in maturing oocytes, we also observed transcription-dependent PSer5 staining in pre-ZGA and mitotic embryonic cells after RNAi knockdown of fcp-1, uba-1, or ubc-2 (). While we cannot rule out that this phosphorylated Pol II simply perdured from oogenesis, it seems more likely that it was produced de novo in these cells because their PSer5 levels were comparable to those seen in transcriptionally active embryonic nuclei, as indicated by immunofluorescence and western blotting assays (; data not shown). We speculate that parallels may exist between how transcription is globally blocked during mitosis and meiosis, related cellular processes that each involve chromosome condensation, nuclear division, and a subsequent rapid reactivation of transcription.
Why would oocyte maturation induce abortive transcription steps? During the transition from oocyte to zygote, the transcription machinery must shift between dramatically different gene expression programs (Baugh et al., 2003
; Seydoux and Fire, 1994
; Zeng and Schultz, 2005
). By initiating early transcription steps at some promoters, oocyte maturation could facilitate their subsequent activation in the embryo. Recent studies suggest precedents for such a model. In mitotic mammalian cells, the hsp70i
gene is “bookmarked” in a transcriptionally competent state, so that it can later be induced more rapidly by stress during G1
(Xing et al., 2005
). Similarly, during stationary phase in S. cerevisiae
Pol II is bound upstream of >2500 genes that are silent, but will be induced within minutes after refeeding (Radonjic et al., 2005
). We speculate that oocyte maturation may similarly poise the Pol II machinery for rapid induction of transcription at some promoters, and that in some animals preparations for ZGA may begin before fertilization. Recent work has shown that maturation signals lead to “marking” of some maternally provided proteins for degradation in the early embryo (Stitzel et al., 2006
). In light of those findings and our results, we believe that maturation signals not only lead to preparations for fertilization, but also may initiate processes that facilitate the transition from maternal to embryonic gene expression.