Academic journal article Genetics

Inhibition of Transcription by the Caenorhabditis Elegans Germline Protein PIE-1: Genetic Evidence for Distinct Mechanisms Targeting Initiation and Elongation

Academic journal article Genetics

Inhibition of Transcription by the Caenorhabditis Elegans Germline Protein PIE-1: Genetic Evidence for Distinct Mechanisms Targeting Initiation and Elongation

Article excerpt

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.

INHIBITION of mRNA transcription is a defining characteristic of the embryonic germ lineage in invertebrates and vertebrates (SEYDOUX and BRAUN 2006). In Drosophila and Caenorhabditis elegans, mRNA synthesis appears to be globally, if not completely, inhibited in the embryonic germ lineage from the onset of embryo-genesis to gastrulation. Early studies in Drosophila embryos showed that somatic nuclei incorporate radio-labeled UTP at a higher rate compared to germline nuclei (ZALOKAR 1976). Expression of the transcriptional activator VP16 could turn on a synthetic target gene in somatic cells but not in germ cells (VAN D OREN et al. 1998). In C. elegans embryos, in situ hybridization experiments using 16 gene-specific probes detected zygotic transcripts in somatic blastomeres, but not in germline blastomeres (SEYDOUX et al. 1996). The only exceptions were ribosomal rRNAs, which appear to be synthesized in both cell types (SEYDOUX and D UNN 1997). Further evidence for a lack of transcription specific to mRNAs was obtained using antibodies against the carboxyl-terminal domain (CTD) of RNA polymer-ase II (SEYDOUX and DUNN 1997; MARTINHO et al. 2004).

The CTD is a long extension of the large subunit of RNA polymerase II containing several (42 in C. elegans and 52 in humans) tandem copies of the heptapeptide motif (Y^sub 1^ S^sub 2^ P^sub 3^T^sub 4^S^sub 5^P^sub 6^S^sub 7^) (PHATNANI and G REENLEAF 2006, for review). The phosphorylation status of the serines in these repeats changes as RNA polymerase II proceeds through the transcription cycle. The repeats start out unphosphorylated as RNA polymerase is recruited into the initiation complex at the promoter. During promoter clearance, Ser5 of each repeat becomes phosphorylated by cyclin-dependent kinase in the TFIIH complex (CDK7), and during the elongation phase, Ser2 becomes phosphorylated by cyclin-dependent kinase in the P-TEFb complex (CDK9). These phosphorylations allow the CTD to function as a scaffold to integrate transcription with processing, including capping, splicing, and termination. Phosphorylation of the CTD occurs in competition with CTD phosphatases to allow unphos-phorylated RNA polymerase II to recycle back into new initiation complexes. Monoclonal antibodies (H14 and H5) that recognize preferentially P-Ser5 or P-Ser2 (PATTURAJAN et al. 1998) have been used widely to characterize the phosphorylation status of the CTD in vivo . Chromatin immunoprecipitation (ChIP) experiments using H14 and H5 have shown that P-Ser5 predominates at the 59 end of genes, whereas P-Ser2 predominates near the 39 end (PHATNANI and GREENLEAF 2006). Immuno-fluorescence studies using these same antibodies in Drosophila and C. elegans embryos have shown that somatic nuclei become positive for P-Ser5 and P-Ser2 coincident with the onset of zygotic transcription. …

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