Drosophila Pgc protein inhibits P-TEFb recruitment to chromatin in primordial germ cells

Abstract

Germ cells are the only cells that transmit genetic information to the next generation, and they therefore must be prevented from differentiating inappropriately into somatic cells. A common mechanism by which germline progenitors are protected from differentiation-inducing signals is a transient and global repression of RNA polymerase II (RNAPII)-dependent transcription. In both Drosophila and Caenorhabditis elegans embryos, the repression of messenger RNA transcription during germ cell specification correlates with an absence of phosphorylation of Ser 2 residues in the carboxy-terminal domain of RNAPII (hereafter called CTD), a critical modification for transcriptional elongation. Here we show that, in Drosophila embryos, a small protein encoded by polar granule component (pgc) is essential for repressing CTD Ser 2 phosphorylation in newly formed pole cells, the germline progenitors. Ectopic Pgc expression in somatic cells is sufficient to repress CTD Ser 2 phosphorylation. Furthermore, Pgc interacts, physically and genetically, with positive transcription elongation factor b (P-TEFb), the CTD Ser 2 kinase complex, and prevents its recruitment to transcription sites. These results indicate that Pgc is a cell-type-specific P-TEFb inhibitor that has a fundamental role in Drosophila germ cell specification. In C. elegans embryos, PIE-1 protein segregates to germline blastomeres, and is thought to repress mRNA transcription through interaction with P-TEFb. Thus, inhibition of P-TEFb is probably a common mechanism during germ cell specification in the disparate organisms C. elegans and Drosophila.

Figure 1. Pgc expression in pole cells is essential for the repression of CTD Ser 2 phosphorylation

a, Pgc sequences of 12 Drosophila species. Conserved amino acid residues are highlighted by green shading. Note that no apparent Pgc orthologues can be found in other animal groups. b, Pgc expression in pole cells is complementary to pSer 2. Panels show the posterior pole of wild-type embryos immunostained for Pgc (green) and pSer 2 (magenta). Nuclei were counter-stained with 4,6-diamidino-2-phenylindole (DAPI; cyan). Pgc is concentrated in the nucleus (inset). c, Posterior poles of stage-4 embryos immunostained for pSer 2 (magenta) and Pgc (green). Nuclei were counter-stained with DAPI. Maternal genotypes are indicated. Note that pgc ORF^fs RNA accumulates normally in the germ plasm (Supplementary Fig. 2).

Figure 2. Pgc is sufficient to repress CTD Ser 2 phosphorylation in somatic cells

Embryos were immunostained for Pgc (green) and pSer 2 (magenta). High magnifications of anterior and posterior parts of the embryos are also shown. To misexpress Pgc at the anterior somatic cell region, a hybrid gene, in which the pgc ORF was fused with the bicoid (bcd) anterior localization signal, was expressed in oogenesis. In embryos expressing pgc-bcd 3′ UTR mRNA, CTD Ser 2 phosphorylation was repressed in regions expressing ectopic Pgc (arrowheads). Many embryos (50–80%) expressing the pgc ORF-bcd 3′ UTR transgene died with variable defects, but we never observed a bicaudal phenotype, suggesting that anterior misexpression of Pgc is incapable of recruiting germ plasm components required for posterior development.

Figure 3. Pgc interacts, physically and genetically, with P-TEFb

a, Cdk9 and CycT, but not Cdk7, were co-immunoprecipitated with Pgc from the lysates of S2 cells expressing 3×Flag–Pgc. b, Lysates of y w or pgc− embryos expressing the flag–pgc transgene were immunoprecipitated with anti-Flag antibody, and bound proteins were analysed by western blotting. c, P-TEFb was overexpressed in pole cells by expressing cycT-nos 3′ UTR and cdk9-nos 3′ UTR transgenes in oogenesis. Overexpression of P-TEFb in pole cells caused precocious CTD pSer 2 phosphorylation (magenta), even in the presence of Pgc expression (green). This precocious CTD pSer 2 phosphorylation was strongly induced when P-TEFb was overexpressed in the pgc^Δ1 heterozygous background.

Figure 4. Pgc prevents P-TEFb recruitment

a, Salivary gland polytene chromosome squashes prepared from third-instar larvae carrying either the sgs3-Gal4 driver alone or sgs3-Gal4 plus two copies of UAST-pgc ORF transgenes immunostained for pSer 5, CycT and Cdk9, and counter-stained with DAPI. Arrowheads indicate P-TEFb signals on cell debris in the Pgcexpressing salivary gland squash. b, Western blot analysis of salivary gland lysates from sgs3-Gal4 or sgs3-Gal4; 2×UAST-Pgc third-instar larvae. c, Real-time PCR analyses of ChIP experiments on the Hsp70 and Hsp27 gene regions in heatshocked (HS) or non-heat-shocked (NHS) S2 cell transfectants. The proportion of Pgc-expressing S2 cells after CuSO₄ induction was about 70%. Each result shows an average of at least three independent experiments with the standard error of the mean.