Inhibition of transcription by the Caenorhabditis elegans germline protein PIE-1: genetic evidence for distinct mechanisms targeting initiation and elongation

Abstract

In Caenorhabditis elegans embryos, specification of the germ lineage depends on PIE-1, a maternal protein that blocks mRNA transcription in germline blastomeres. Studies in mammalian cell culture have suggested that PIE-1 inhibits P-TEFb, a kinase that phosphorylates serine 2 in the carboxyl-terminal domain (CTD) repeats of RNA polymerase II during transcriptional elongation. We have tested this hypothesis using an in vivo complementation assay for PIE-1 function. Our results support the view that PIE-1 inhibits P-TEFb using the CTD-like motif YAPMAPT. This activity is required to block serine 2 phosphorylation in germline blastomeres, but unexpectedly is not essential for transcriptional repression or specification of the germline. We find that sequences outside of the YAPMAPT are required to inhibit serine 5 phosphorylation, and that this second inhibitory mechanism is essential for transcriptional repression and specification of the germ lineage. Our results suggest that PIE-1 uses partially redundant mechanisms to block transcription by targeting both the initiation and elongation phases of the transcription cycle.

Figure 1.—

PIE-1 binds to CIT-1.1 in vitro. (A) In vitro translated and ³⁵S-labeled full-length PIE-1 and mutant derivatives (section 1—input) were incubated with immobilized MBP:CIT-1.1 (section 2) or negative control MBP:PAR-5 (section 3) and bound proteins were resolved by SDS–PAGE. Sections 4 and 5 show Coomassie staining of MBP:CIT-1.1 (section 4) and MBP:PAR-5 (section 5) to control for loading. ELN-C is elongin C (DeRenzo et al. 2003) used here as a negative control. Numbers below sections 2 and 3 indicate percentage bound (bound/input × 100%), as calculated by measuring band intensities using Imagequant software (Molecular Dynamics). (B) Diagram showing the sequence of the C-terminal domain of PIE-1 and the mutant derivatives used in this study. The minimal repressor domain is the minimal PIE-1 fragment that can inhibit transcription when artificially brought to a promoter in HeLa cells (Batchelder et al. 1999).

Figure 1.—

PIE-1 binds to CIT-1.1 in vitro. (A) In vitro translated and ³⁵S-labeled full-length PIE-1 and mutant derivatives (section 1—input) were incubated with immobilized MBP:CIT-1.1 (section 2) or negative control MBP:PAR-5 (section 3) and bound proteins were resolved by SDS–PAGE. Sections 4 and 5 show Coomassie staining of MBP:CIT-1.1 (section 4) and MBP:PAR-5 (section 5) to control for loading. ELN-C is elongin C (DeRenzo et al. 2003) used here as a negative control. Numbers below sections 2 and 3 indicate percentage bound (bound/input × 100%), as calculated by measuring band intensities using Imagequant software (Molecular Dynamics). (B) Diagram showing the sequence of the C-terminal domain of PIE-1 and the mutant derivatives used in this study. The minimal repressor domain is the minimal PIE-1 fragment that can inhibit transcription when artificially brought to a promoter in HeLa cells (Batchelder et al. 1999).

Figure 2.—

Inhibition of P-Ser5 and P-Ser2 by the PIE-1 transgenes. (A) Eight- to 15-cell pie-1(zu127) embryos expressing the indicated PIE-1 transgenes and stained for DAPI and P-Ser2 or P-Ser5. Arrow points to the germline blastomeres. (B) Close-up of germline blastomere nuclei stained for P-Ser2 or P-Ser5 in pie-1(zu127) embryos expressing the indicating PIE-1 transgenes.

Figure 3.—

Wild-type PIE-1 and PIE-1(DAQMEQT) inhibit transcription of a pes-10:gfp transgene. In situ hybridization shows nuclear accumulation of zygotic pes-10:gfp RNA in pie-1(zu127) embryos expressing the indicated transgenes. Arrows point to germline blastomeres, which do not accumulate pes-10:gfp RNA. Fourteen of 14 embryos expressing wild-type PIE-1 and 28/28 embryos expressing PIE-1(DAQMEQT) showed this pattern.

Figure 4.—

Western blot of GFP:PIE-1 fusions. Total worm extracts from strains expressing the indicated PIE-1 fusions were immunoblotted with anti-GFP antibody. Anti-tubulin is used as the loading control.

Figure 5.—

GFP:PIE-1 localization in nuclei. Collapsed confocal Z-stacks through the P₂ germline blastomere show accumulation of GFP:PIE-1 in nuclei. Wild-type PIE-1 accumulates in numerous fine nuclear foci. This localization is disrupted in PIE-1 mutants that do not suppress P-Ser5 efficiently (marked with asterisk). Reductions in CDK-7, CIT-1.1/1.2, and AMA-1 (RNA polymerase II) levels also affect this localization. Note that PIE-1 also accumulates in larger cytoplasmic granules (P granules), which are visible around the nuclei in the micrographs.