Throughout embryonic development, transcriptional repression of the primordial germ cells is a fundamental means of maintaining germ cell fate by preventing activation of somatic programs. In C. elegans, germline blastomeres are initially kept transcriptionally quiescent by the maternally loaded CCCH zinc-finger protein PIE-1, which is thought to inhibit transcriptional elongation in the P lineage (Seydoux et al., 1996; Zhang et al., 2003). PIE-1 disappears upon the birth of the primordial germline precursor cells Z2 and Z3, yet these cells still appear to remain quiescent. We have previously demonstrated that there is a chromatin-based repressive mechanism that succeeds PIE-1 degradation by which the chromatin in Z2/Z3 globally loses certain histone modifications including histone H3 lysine 4 dimethylation (H3K4me2), a conserved marker for transcriptionally competent chromatin (Schaner et al., 2003) The mechanistic details of this global remodeling event, however, remain to be elucidated. Upon the birth of Z2/Z3 and the disappearance of PIE-1, RNA polymerase II phosphorylated on serine 2 of the C-terminal domain repeat (Pol II
ser2-P) appears in the PGCs (Seydoux and Dunn, 1997). Here we show that in the absence of RNA polymerase II (Pol II) transcription, embryos fail to lose H3K4me2 in Z2/Z3, indicating that Pol II is essential for global chromatin remodeling in Z2/Z3. Furthermore, failure to remodel chromatin in Z2/Z3 is also observed in the absence of either of two kinases that may each have roles in
ser2-P regulation,
cdk-9 and
tlk-1. In
cdk-9 (RNAi);
tlk-1(RNAi) embryos,
ser2-P staining is completely diminished from Z2/Z3, suggesting a putative connection between phosphorylation states of Pol II and onset of chromatin remodeling. We have also observed this phenotype in the absence of SKP-1, a highly conserved protein that plays dual roles as both a transcriptional co-factor and essential spliceosome component.
skp-1 (RNAi) embryos fail to lose H3K4me2 in Z2/Z3, and although Pol II is present on all nuclei,
ser2-P levels are significantly reduced in all cells. It is currently unknown, however, whether transcription is either a direct or indirect requirement for Z2/Z3 chromatin remodeling. We are currently assessing the roles of translational machinery and spliceosome components in these processes. We are also continuing to investigate the link between PIE-1 degradation, the onset of chromatin remodeling, and the appearance of
ser2-P in Z2/Z3.