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Repression of early zygotic transcription in the germline
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Repression of early zygotic transcription in the germline Akira Nakamura, Maki Shirae-Kurabayashi and Kazuko Hanyu-Nakamura Germ cells, the progenitors of gametes, are often specified and segregated from somatic lineages early in embryogenesis. As germ cells are essential to create the next generation in sexually reproducing organisms, they must be prevented from differentiating inappropriately into somatic cells. In Drosophila and Caenorhabditis elegans embryos, this is governed by the transient and global repression of mRNA transcription. Furthermore, the inhibition of somatic transcriptional programs is also crucial for germ cell specification in the mouse. Therefore, the active repression of somatic transcriptional programs appears to be a common mechanism for launching the germline. In this review, we will discuss the mechanisms of transcriptional repression during germ cell specification and their interspecies similarities and differences. Address Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan Corresponding author: Nakamura, Akira ([email protected])
Current Opinion in Cell Biology 2010, 22:709–714 This review comes from a themed issue on Cell differentiation Edited by Mark Van Doren
0955-0674/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2010.08.012
Introduction Multicellular organisms are composed of two major cell types: somatic and germline. Somatic cells differentiate into many cell types that are organized into tissues and organs. Somatic cells are therefore vital for individual lives, but their functions cease upon the organism’s death. By contrast, germ cells, the progenitors of gametes, create the next generation of the species, through fertilization. As germ cells are essential for the transmission of genetic information to the next generation, only mutations that occur in the germline genome can be transmitted to the progeny. Therefore, germ cells also play important roles in evolution. Germ cells often segregate from somatic lineages during early embryogenesis [1]. Two modes of germ cell formation are known [2]. One is epigenetic specification, in which germ cells are induced among pluripotent cell populations by extracellular signals. The other is preformation, in which germ cell formation www.sciencedirect.com
depends on maternal factors located in the germ plasm, a specialized cytoplasmic region within the egg. Although the epigenetic mode of germ cell formation appears to be general from an evo devo point of view [2], many model organisms, such as Drosophila, Caenorhabditis elegans, Xenopus, and Ciona, deploy the preformation mode of germ cell specification with the germ plasm in the egg (Figure 1). One of the key functions of the germ plasm is to protect germ cell progenitors from somatic differentiation. In Drosophila and C. elegans, this process is governed at the level of transcription [3]. Germ cell progenitors in both organisms display a global repression of mRNA transcription until the germ cell fate is fully established. Recent studies in mouse have revealed that the selective inhibition of somatic transcriptional programs is also crucial for the epigenetic specification of germ cells in this animal [4,5–7].
Germ cell formation in Drosophila and C. elegans The germ plasm of the Drosophila egg, also called the pole plasm, is sequestered in the posterior pole region, in which many maternal mRNAs and proteins are localized and stably anchored [8]. The early Drosophila embryo develops in a syncytium, in which nuclei multiply in the middle of the embryo and then start moving to the periphery at the seventh division. The majority of the nuclei arriving at the periphery form a layer of somatic cells, but several nuclei that reach the posterior pole pinch off with the pole plasm to form pole cells, the germ cell progenitors. The pole plasm contains sufficient factors to confer the germline fate on the undetermined nuclei, since the transplantation or mis-assembly of the germ plasm at the anterior region of the egg induces ectopic pole cells [9,10]. During gastrulation, about 40 pole cells are carried to the interior of the embryo, surrounded by the midgut epithelium. They then migrate across the midgut cell layer into the body cavity, to the mesoderm, where they are ensheathed by somatic gonadal precursors to form a pair of embryonic gonads [11]. In late embryogenesis, the pole cells resume mitosis within the gonads, where a small portion of the pole cells become germline stem cells to ensure the continuous production of gametes in adults [12]. The segregation of the germline from the soma in C. elegans is established during the first four rounds of asymmetric cell divisions after the egg is fertilized. The C. elegans egg contains granular structures, called P-granules, which consist of specific sets of maternal RNAs and proteins [13]. They are initially distributed uniformly throughout the cytoplasm of the unfertilized egg. Upon fertilization, the Current Opinion in Cell Biology 2010, 22:709–714
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Figure 1
Early germ cell development in several model animals. (a) The Drosophila germ plasm, also called the pole plasm (pink), is assembled in the posterior pole of the egg. The pole plasm is incorporated into pole cells, which are carried to the interior of the embryo during gastrulation, before becoming migratory. The pole cells then actively cross the midgut epithelium to enter the body cavity, where they attach to somatic gonadal precursors to form embryonic gonads. (b) The germ plasm of C. elegans (pink) represents as P-granules, which are uniformly distributed in the unfertilized egg. Upon fertilization, the P-granules move toward the posterior and are incorporated into the P1 blastomere. Through additional asymmetric divisions, Pgranules are partitioned into the germline blastomere, P4, which divides once at the 100-cell stage to produce the PGCs, Z2 and Z3. (c) The germ plasm of Xenopus (pink) is sequestered at the vegetal pole of the egg. It is segregated unequally during the cleavage stage. Cells incorporating the germ plasm are the primordial germ cells, which remain in the endoderm during gastrulation, proliferate, and then migrate dorsally through the lateral endoderm during the tailbud stage, until they reach the genital ridges in the larvae. (d) The germ plasm of ascidians is thought to reside in the postplasm, which accumulates at the posterior pole of the egg upon fertilization (pink). The postplasm is segregated into the posterior-most pair of blastomeres during the cleavage stages, and is partitioned into the pair of B7.6 cells in the 110-cell-stage embryo. B7.6 cells undergo an asymmetric division during gastrulation to produce two distinct cells, the B8.11 and B8.12 cells, which are carried in the tail region of the tailbud embryo. Lineage tracing experiments have revealed that B8.12 cells but not B8.11 cells develop into PGCs after metamorphosis [41]. (e) During E6.25–6.5 of mouse embryogenesis, signals from extraembryonic ectoderm promote a few proximal epiblast cells to express Blimp1. These Blimp1-expressing cells proliferate and migrate to the posterior to become 40 stella-positive and AP-positive definitive PGCs, which are present in the extraembryonic mesoderm posterior to the primitive streak at E7.25–7.5, and migrate back to the embryo thereafter. By E11.5, the PGCs reach and enter the genital ridges, where they undergo sexual differentiation according to the somatic sex of the embryo.
P-granules move toward the posterior region of the embryo, causing the asymmetric partition of the P-granules to the P1 germline blastomere. Another blastomere, called AB, does not incorporate P-granules and cannot generate germ cells. In the subsequent divisions, the P-granules are inherited only by the germline blastomeres and are eventually partitioned into a single blastomere, P4. At the 100cell stage, P4 undergoes a symmetric division to form Current Opinion in Cell Biology 2010, 22:709–714
equivalent primordial germ cells (PGCs), Z2 and Z3, which later resume mitosis, expanding their number before their entry into meiosis, thus allowing the production of a large number of gametes in the adult.
Transcriptional quiescence in the germline Evidence for transcriptional silencing in newly formed germ cells can be traced back to the work performed in www.sciencedirect.com
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the 1970s [14]. In this study, the de novo syntheses of RNA and protein in early Drosophila embryos were traced with radiolabeled RNA or protein precursors. The results of these experiments showed that the somatic cells of cellular-blastoderm-stage embryos incorporated a significant amount of [3H]-uridine (an RNA precursor), but no tritium radioactivity was observed in the pole cells until they started migrating across the midgut. In contrast, the de novo synthesis of proteins was readily detected in the pole cells. A lack of transcriptional activators was shown to be an unlikely cause of transcriptional quiescence in the early pole cells, since the introduction of an artificial transcriptional activator, GAL4VP16, did not drive mRNA production in them [15]. In C. elegans embryos, active mRNA transcription is detectable from the 4-cell stage onward in all somatic blastomeres that do not generate germ cells [16]. By contrast, the zygotic mRNA transcription in the germline is first detectable after the P4 blastomere divides to form the Z2 and Z3 cells. Thus, the onset of mRNA transcription is delayed in the C. elegans germline until the germ cell fate is fully established. In both Drosophila and C. elegans embryos, the transcription of ribosomal RNAs is detectable in germ cell precursors [3]. Therefore, RNA polymerase II (RNAPII)-dependent transcription appears to be specifically and globally blocked during germ cell specification in both animals. However, the question of how the RNAPII-dependent transcription is repressed in the germ cells of these animals remains unanswered.
Repression of RNAPII phosphorylation in the germline The largest subunit of RNAPII contains a specialized carboxy-terminal domain (CTD) that consists of a repeated heptapeptide sequence, YSPTSPS (26 repeats in Saccharomyces cerevisiae, 42 in Drosophila and C. elegans, and 52 in human). During transcription cycles, the serine residues at the second and fifth positions (hereafter referred to as Ser2 or Ser5, respectively) in each repeat undergo extensive phosphorylation and dephosphorylation, providing a platform for the recruitment and assembly of factors involved in mRNA transcription and processing [17]. In Drosophila embryos, although both phosphorylated Ser2 and Ser5 (referred to as pSer2 or pSer5, respectively) are abundant in all somatic nuclei from the cellular blastoderm stage onward, pSer5 is reduced and pSer2 is undetectable in the pole cells before gastrulation [3]. Similarly, in C. elegans embryos, low pSer5 and no pSer2 are detectable in the P1–P4 germline blastomeres. Biochemical studies have revealed that RNAPII CTD Ser5 phosphorylation correlates with transcription initiation and promoter clearance, whereas Ser2 phosphorylation is crucial for coupling splicing and 30 end processing to promote transcriptional elongation [17,18]. Thus, the transient lack of pSer2 in the germline suggests www.sciencedirect.com
that the mRNA transcription in germ cells could be blocked at the elongation step.
PIE-1 represses germline transcription in the C. elegans embryo C. elegans pie-1 was first isolated as a maternal-effect mutant, in which the P2 germline blastomere develops to resemble its sister blastomere, EMS [16,19]. In wildtype embryos, the maternally supplied transcription factor SKN-1 is partitioned into both the EMS and P2 blastomeres, but it is only active in EMS due to the transcriptional quiescence of P2. In embryos lacking pie-1, however, the P2 blastomere is transcriptionally active and expresses SKN-1’s target genes, which switches the P2 fate to an EMS-like lineage. Consistent with this, high levels of pSer2 and pSer5 are detected in the P2 blastomere in pie-1 embryos [3]. PIE-1 accumulates in the nucleus as well as in P-granules in the P1–P4 blastomeres during embryogenesis. In addition, the ectopic expression of PIE-1 in C. elegans embryos is sufficient to repress mRNA transcription in somatic blastomeres. The precise mechanisms by which PIE-1 represses mRNA transcription remain unsolved. The tethering of PIE-1 to a promoter represses reporter mRNA transcription in human cells, suggesting that PIE-1 interferes directly with the RNAPII-dependent transcriptional machinery [20]. Notably, PIE-1 binds CyclinT (CycT), a component of the positive transcriptional elongation factor b (P-TEFb), which binds CTD and phosphorylates Ser2 to promote transcriptional elongation [21]. PIE-1 interacts with CycT through a YAPMAPT amino acid sequence, which resembles a non-phosphorylatable version of the CTD heptapeptide (YSPTSPS). This suggests that PIE-1 competes with the RNAPII CTD for the interaction with P-TEFb [20]. Interestingly, genetic analyses suggest that PIE-1 has additional, P-TEFbindependent mechanism to repress mRNA transcription [22].
Pgc acts as a P-TEFb inhibitor in Drosophila pole cells Similar to the repression of mRNA transcription during germ cell specification in C. elegans, that in Drosophila is achieved by inhibiting P-TEFb action. In this process, polar granule component ( pgc) plays an essential role [23]. Originally, pgc was identified as a maternal RNA that is enriched in the pole plasm [24]. In embryos lacking pgc, pole cells form normally. However, they degenerate during gastrulation, and few pole cells coalesce with the embryonic gonads. As a result, most of these flies become sterile adults [24]. Pole cells lacking pgc fail to downregulate CTD Ser2 phosphorylation, and misexpress several somatic genes that are normally expressed only in neighboring somatic cells [23,25,26]. Therefore, pgc is the Drosophila germ plasm factor that represses the mRNA transcription in newly formed pole cells. pgc Current Opinion in Cell Biology 2010, 22:709–714
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encodes a short, 71-amino-acid protein, which is conserved only among Drosophila species [23,27]. Pgc protein is transiently expressed in pole cells, in a manner that is temporally reciprocal to CTD Ser2 phosphorylation [23,28]. Furthermore, the misexpression of Pgc in the anterior pole of the embryo as well as in the Drosophila S2 cell line suppresses the phosphorylation of Ser2 of RNAPII CTD, showing that Pgc is sufficient to downregulate pSer2, even in somatic cells [23,27]. Given that Pgc can function in somatic cells, it seemed likely that it interacts with the general machinery of RNAPII-dependent transcription. As expected, the overexpression of P-TEFb in pole cells was found to promote precocious CTD Ser2 phosphorylation even in the presence of Pgc, and to cause pole cell loss during gastrulation [23]. These observations suggest that Pgc interferes with P-TEFb to confer transcriptional quiescence on newly formed pole cells. Indeed, Pgc was found to form a complex with P-TEFb through interaction with the Cdk9 subunit. Pgc alone cannot inhibit P-TEFb’s phosphorylation of CTD Ser2 in vitro. However, the ectopic expression of Pgc in salivary glands sequesters the P-TEFb away from chromosomes. Consistent with this, a chromatin immunoprecipitation (ChIP) assay in S2 cells showed that Pgc prevents the recruitment of P-TEFb to transcriptional sites. Taken together, these data support the idea that Pgc is a P-TEFb inhibitor that plays a fundamental role in Drosophila germ cell specification [23].
Interspecies similarities in establishing germline transcriptional quiescence PIE-1 and Pgc are unique to nemotodes and Drosophila, respectively, and they therefore must have arisen independently in evolution. Nevertheless, both PIE-1 and Pgc repress mRNA transcription between its initiation and elongation steps by inhibiting P-TEFb. This suggests that elongation is an important regulatory point in RNAPIIdependent transcription. Intriguingly, recent genome-wide analyses using ChIP and whole-genome tiling arrays (ChIP–chip) in yeast, human, and Drosophila showed that the post-recruitment regulation of RNAPII-dependent transcription is much more general than previously thought [29–33]. Furthermore, the activation of transcription in mouse embryonic stem cells is generally accomplished with the release of RNAPII pausing, presumably through the recruitment of P-TEFb by the transcription factor cMyc [34]. Since germ cells begin their own transcriptional program later in development, stalling the RNAPII function by inhibiting P-TEFb may be an efficient mechanism for establishing transient transcriptional quiescence without losing transcriptional competence.
Silencing of somatic transcriptional programs also occurs in the mouse germline Unlike in Drosophila and C. elegans, the germ cells in mouse are not pre-determined by the germ plasm, but Current Opinion in Cell Biology 2010, 22:709–714
instead arise through an epigenetic mechanism in response to signals from the extraembryonic ectoderm [35,36]. PGCs in the mouse emerge as 40 alkaline phosphatase (AP)-positive and stella-positive cells that form a cluster posterior to the primitive streak in the extraembryonic mesoderm at embryonic day (E) 7.25 [6,37]. Earlier in embryogenesis, the PGC precursors reside within the proximal epiblast cells. The transcriptional regulator Blimp1 plays key roles in PGC specification [5]. Blimp1 expression is induced only in a few cells among the proximal posterior epiblast cells at E6.25–6.5, by signals from extracellular tissues [35]. These cells proliferate and contribute almost invariably to the stella-positive PGCs. In Blimp1-deficient embryos, the PGC precursors fail to migrate to and populate the embryonic gonads. Mouse PGCs arise from cells that are otherwise destined to become mesoderm, and they express a number of genes involved in mesodermal development, such as Hox, Snail, and Tbx [4,5,7]. These mesodermal genes are downregulated in Blimp1-positive PGC precursors, which in turn upregulate the expression of genes required for pluripotency (e.g. Sox2 and Nanog) as well as germ cell development (e.g. Nanos3 and Dnd1). Notably, transcriptional profiling using highly representative single-cell microarray technology indicates that Blimp1 is responsible for repressing virtually all of the transcripts that are downregulated during PGC specification [4,38]. Unlike PIE-1 in C. elegans and Pgc in Drosophila, mouse Blimp1 does not globally repress mRNA transcription during PGC specification. Nevertheless, the repression of somatic transcriptional programs is an essential process in launching the germline in mouse.
Conclusion Germline transcriptional repression has been observed in a number of animal embryos. As in Drosophila and C. elegans, PGCs in Xenopus embryos show a transient and specific repression of CTD Ser2 phosphorylation [39], although the factor responsible for the process remains unknown. Zygotic transcription in the germline appears to be repressed in ascidian embryos as well. While no downregulation of CTD phosphorylation in the germline has been detected in Halocynthia roretzi embryos [40], germline blastomeres in Ciona intestinalis embryos show a marked reduction in pSer2 signals during the cleavage stage (M.S.K., unpublished observations). Although distinct mechanisms and factors are responsible for transcriptional quiescence in different organisms, the active repression of somatic transcriptional programs during the establishment of the germline is widespread in animal development, regardless of the mode of germ cell formation.
Acknowledgements The authors thank H. Sano and M. Van Doren for comments on the manuscript. Research in our laboratory is supported in part by a Grant-inAid from the Ministry of Education, Culture, Sports and Science, Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS). www.sciencedirect.com
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Repression of early zygotic transcription in the germline Akira Nakamura, Maki Shirae-Kurabayashi and Kazuko Hanyu-Nakamura Germ cells, the progenitors of gametes, are often specified and segregated from somatic lineages early in embryogenesis. As germ cells are essential to create the next generation in sexually reproducing organisms, they must be prevented from differentiating inappropriately into somatic cells. In Drosophila and Caenorhabditis elegans embryos, this is governed by the transient and global repression of mRNA transcription. Furthermore, the inhibition of somatic transcriptional programs is also crucial for germ cell specification in the mouse. Therefore, the active repression of somatic transcriptional programs appears to be a common mechanism for launching the germline. In this review, we will discuss the mechanisms of transcriptional repression during germ cell specification and their interspecies similarities and differences. Address Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan Corresponding author: Nakamura, Akira ([email protected])
Current Opinion in Cell Biology 2010, 22:709–714 This review comes from a themed issue on Cell differentiation Edited by Mark Van Doren
0955-0674/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2010.08.012
Introduction Multicellular organisms are composed of two major cell types: somatic and germline. Somatic cells differentiate into many cell types that are organized into tissues and organs. Somatic cells are therefore vital for individual lives, but their functions cease upon the organism’s death. By contrast, germ cells, the progenitors of gametes, create the next generation of the species, through fertilization. As germ cells are essential for the transmission of genetic information to the next generation, only mutations that occur in the germline genome can be transmitted to the progeny. Therefore, germ cells also play important roles in evolution. Germ cells often segregate from somatic lineages during early embryogenesis [1]. Two modes of germ cell formation are known [2]. One is epigenetic specification, in which germ cells are induced among pluripotent cell populations by extracellular signals. The other is preformation, in which germ cell formation www.sciencedirect.com
depends on maternal factors located in the germ plasm, a specialized cytoplasmic region within the egg. Although the epigenetic mode of germ cell formation appears to be general from an evo devo point of view [2], many model organisms, such as Drosophila, Caenorhabditis elegans, Xenopus, and Ciona, deploy the preformation mode of germ cell specification with the germ plasm in the egg (Figure 1). One of the key functions of the germ plasm is to protect germ cell progenitors from somatic differentiation. In Drosophila and C. elegans, this process is governed at the level of transcription [3]. Germ cell progenitors in both organisms display a global repression of mRNA transcription until the germ cell fate is fully established. Recent studies in mouse have revealed that the selective inhibition of somatic transcriptional programs is also crucial for the epigenetic specification of germ cells in this animal [4,5–7].
Germ cell formation in Drosophila and C. elegans The germ plasm of the Drosophila egg, also called the pole plasm, is sequestered in the posterior pole region, in which many maternal mRNAs and proteins are localized and stably anchored [8]. The early Drosophila embryo develops in a syncytium, in which nuclei multiply in the middle of the embryo and then start moving to the periphery at the seventh division. The majority of the nuclei arriving at the periphery form a layer of somatic cells, but several nuclei that reach the posterior pole pinch off with the pole plasm to form pole cells, the germ cell progenitors. The pole plasm contains sufficient factors to confer the germline fate on the undetermined nuclei, since the transplantation or mis-assembly of the germ plasm at the anterior region of the egg induces ectopic pole cells [9,10]. During gastrulation, about 40 pole cells are carried to the interior of the embryo, surrounded by the midgut epithelium. They then migrate across the midgut cell layer into the body cavity, to the mesoderm, where they are ensheathed by somatic gonadal precursors to form a pair of embryonic gonads [11]. In late embryogenesis, the pole cells resume mitosis within the gonads, where a small portion of the pole cells become germline stem cells to ensure the continuous production of gametes in adults [12]. The segregation of the germline from the soma in C. elegans is established during the first four rounds of asymmetric cell divisions after the egg is fertilized. The C. elegans egg contains granular structures, called P-granules, which consist of specific sets of maternal RNAs and proteins [13]. They are initially distributed uniformly throughout the cytoplasm of the unfertilized egg. Upon fertilization, the Current Opinion in Cell Biology 2010, 22:709–714
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Figure 1
Early germ cell development in several model animals. (a) The Drosophila germ plasm, also called the pole plasm (pink), is assembled in the posterior pole of the egg. The pole plasm is incorporated into pole cells, which are carried to the interior of the embryo during gastrulation, before becoming migratory. The pole cells then actively cross the midgut epithelium to enter the body cavity, where they attach to somatic gonadal precursors to form embryonic gonads. (b) The germ plasm of C. elegans (pink) represents as P-granules, which are uniformly distributed in the unfertilized egg. Upon fertilization, the P-granules move toward the posterior and are incorporated into the P1 blastomere. Through additional asymmetric divisions, Pgranules are partitioned into the germline blastomere, P4, which divides once at the 100-cell stage to produce the PGCs, Z2 and Z3. (c) The germ plasm of Xenopus (pink) is sequestered at the vegetal pole of the egg. It is segregated unequally during the cleavage stage. Cells incorporating the germ plasm are the primordial germ cells, which remain in the endoderm during gastrulation, proliferate, and then migrate dorsally through the lateral endoderm during the tailbud stage, until they reach the genital ridges in the larvae. (d) The germ plasm of ascidians is thought to reside in the postplasm, which accumulates at the posterior pole of the egg upon fertilization (pink). The postplasm is segregated into the posterior-most pair of blastomeres during the cleavage stages, and is partitioned into the pair of B7.6 cells in the 110-cell-stage embryo. B7.6 cells undergo an asymmetric division during gastrulation to produce two distinct cells, the B8.11 and B8.12 cells, which are carried in the tail region of the tailbud embryo. Lineage tracing experiments have revealed that B8.12 cells but not B8.11 cells develop into PGCs after metamorphosis [41]. (e) During E6.25–6.5 of mouse embryogenesis, signals from extraembryonic ectoderm promote a few proximal epiblast cells to express Blimp1. These Blimp1-expressing cells proliferate and migrate to the posterior to become 40 stella-positive and AP-positive definitive PGCs, which are present in the extraembryonic mesoderm posterior to the primitive streak at E7.25–7.5, and migrate back to the embryo thereafter. By E11.5, the PGCs reach and enter the genital ridges, where they undergo sexual differentiation according to the somatic sex of the embryo.
P-granules move toward the posterior region of the embryo, causing the asymmetric partition of the P-granules to the P1 germline blastomere. Another blastomere, called AB, does not incorporate P-granules and cannot generate germ cells. In the subsequent divisions, the P-granules are inherited only by the germline blastomeres and are eventually partitioned into a single blastomere, P4. At the 100cell stage, P4 undergoes a symmetric division to form Current Opinion in Cell Biology 2010, 22:709–714
equivalent primordial germ cells (PGCs), Z2 and Z3, which later resume mitosis, expanding their number before their entry into meiosis, thus allowing the production of a large number of gametes in the adult.
Transcriptional quiescence in the germline Evidence for transcriptional silencing in newly formed germ cells can be traced back to the work performed in www.sciencedirect.com
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the 1970s [14]. In this study, the de novo syntheses of RNA and protein in early Drosophila embryos were traced with radiolabeled RNA or protein precursors. The results of these experiments showed that the somatic cells of cellular-blastoderm-stage embryos incorporated a significant amount of [3H]-uridine (an RNA precursor), but no tritium radioactivity was observed in the pole cells until they started migrating across the midgut. In contrast, the de novo synthesis of proteins was readily detected in the pole cells. A lack of transcriptional activators was shown to be an unlikely cause of transcriptional quiescence in the early pole cells, since the introduction of an artificial transcriptional activator, GAL4VP16, did not drive mRNA production in them [15]. In C. elegans embryos, active mRNA transcription is detectable from the 4-cell stage onward in all somatic blastomeres that do not generate germ cells [16]. By contrast, the zygotic mRNA transcription in the germline is first detectable after the P4 blastomere divides to form the Z2 and Z3 cells. Thus, the onset of mRNA transcription is delayed in the C. elegans germline until the germ cell fate is fully established. In both Drosophila and C. elegans embryos, the transcription of ribosomal RNAs is detectable in germ cell precursors [3]. Therefore, RNA polymerase II (RNAPII)-dependent transcription appears to be specifically and globally blocked during germ cell specification in both animals. However, the question of how the RNAPII-dependent transcription is repressed in the germ cells of these animals remains unanswered.
Repression of RNAPII phosphorylation in the germline The largest subunit of RNAPII contains a specialized carboxy-terminal domain (CTD) that consists of a repeated heptapeptide sequence, YSPTSPS (26 repeats in Saccharomyces cerevisiae, 42 in Drosophila and C. elegans, and 52 in human). During transcription cycles, the serine residues at the second and fifth positions (hereafter referred to as Ser2 or Ser5, respectively) in each repeat undergo extensive phosphorylation and dephosphorylation, providing a platform for the recruitment and assembly of factors involved in mRNA transcription and processing [17]. In Drosophila embryos, although both phosphorylated Ser2 and Ser5 (referred to as pSer2 or pSer5, respectively) are abundant in all somatic nuclei from the cellular blastoderm stage onward, pSer5 is reduced and pSer2 is undetectable in the pole cells before gastrulation [3]. Similarly, in C. elegans embryos, low pSer5 and no pSer2 are detectable in the P1–P4 germline blastomeres. Biochemical studies have revealed that RNAPII CTD Ser5 phosphorylation correlates with transcription initiation and promoter clearance, whereas Ser2 phosphorylation is crucial for coupling splicing and 30 end processing to promote transcriptional elongation [17,18]. Thus, the transient lack of pSer2 in the germline suggests www.sciencedirect.com
that the mRNA transcription in germ cells could be blocked at the elongation step.
PIE-1 represses germline transcription in the C. elegans embryo C. elegans pie-1 was first isolated as a maternal-effect mutant, in which the P2 germline blastomere develops to resemble its sister blastomere, EMS [16,19]. In wildtype embryos, the maternally supplied transcription factor SKN-1 is partitioned into both the EMS and P2 blastomeres, but it is only active in EMS due to the transcriptional quiescence of P2. In embryos lacking pie-1, however, the P2 blastomere is transcriptionally active and expresses SKN-1’s target genes, which switches the P2 fate to an EMS-like lineage. Consistent with this, high levels of pSer2 and pSer5 are detected in the P2 blastomere in pie-1 embryos [3]. PIE-1 accumulates in the nucleus as well as in P-granules in the P1–P4 blastomeres during embryogenesis. In addition, the ectopic expression of PIE-1 in C. elegans embryos is sufficient to repress mRNA transcription in somatic blastomeres. The precise mechanisms by which PIE-1 represses mRNA transcription remain unsolved. The tethering of PIE-1 to a promoter represses reporter mRNA transcription in human cells, suggesting that PIE-1 interferes directly with the RNAPII-dependent transcriptional machinery [20]. Notably, PIE-1 binds CyclinT (CycT), a component of the positive transcriptional elongation factor b (P-TEFb), which binds CTD and phosphorylates Ser2 to promote transcriptional elongation [21]. PIE-1 interacts with CycT through a YAPMAPT amino acid sequence, which resembles a non-phosphorylatable version of the CTD heptapeptide (YSPTSPS). This suggests that PIE-1 competes with the RNAPII CTD for the interaction with P-TEFb [20]. Interestingly, genetic analyses suggest that PIE-1 has additional, P-TEFbindependent mechanism to repress mRNA transcription [22].
Pgc acts as a P-TEFb inhibitor in Drosophila pole cells Similar to the repression of mRNA transcription during germ cell specification in C. elegans, that in Drosophila is achieved by inhibiting P-TEFb action. In this process, polar granule component ( pgc) plays an essential role [23]. Originally, pgc was identified as a maternal RNA that is enriched in the pole plasm [24]. In embryos lacking pgc, pole cells form normally. However, they degenerate during gastrulation, and few pole cells coalesce with the embryonic gonads. As a result, most of these flies become sterile adults [24]. Pole cells lacking pgc fail to downregulate CTD Ser2 phosphorylation, and misexpress several somatic genes that are normally expressed only in neighboring somatic cells [23,25,26]. Therefore, pgc is the Drosophila germ plasm factor that represses the mRNA transcription in newly formed pole cells. pgc Current Opinion in Cell Biology 2010, 22:709–714
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encodes a short, 71-amino-acid protein, which is conserved only among Drosophila species [23,27]. Pgc protein is transiently expressed in pole cells, in a manner that is temporally reciprocal to CTD Ser2 phosphorylation [23,28]. Furthermore, the misexpression of Pgc in the anterior pole of the embryo as well as in the Drosophila S2 cell line suppresses the phosphorylation of Ser2 of RNAPII CTD, showing that Pgc is sufficient to downregulate pSer2, even in somatic cells [23,27]. Given that Pgc can function in somatic cells, it seemed likely that it interacts with the general machinery of RNAPII-dependent transcription. As expected, the overexpression of P-TEFb in pole cells was found to promote precocious CTD Ser2 phosphorylation even in the presence of Pgc, and to cause pole cell loss during gastrulation [23]. These observations suggest that Pgc interferes with P-TEFb to confer transcriptional quiescence on newly formed pole cells. Indeed, Pgc was found to form a complex with P-TEFb through interaction with the Cdk9 subunit. Pgc alone cannot inhibit P-TEFb’s phosphorylation of CTD Ser2 in vitro. However, the ectopic expression of Pgc in salivary glands sequesters the P-TEFb away from chromosomes. Consistent with this, a chromatin immunoprecipitation (ChIP) assay in S2 cells showed that Pgc prevents the recruitment of P-TEFb to transcriptional sites. Taken together, these data support the idea that Pgc is a P-TEFb inhibitor that plays a fundamental role in Drosophila germ cell specification [23].
Interspecies similarities in establishing germline transcriptional quiescence PIE-1 and Pgc are unique to nemotodes and Drosophila, respectively, and they therefore must have arisen independently in evolution. Nevertheless, both PIE-1 and Pgc repress mRNA transcription between its initiation and elongation steps by inhibiting P-TEFb. This suggests that elongation is an important regulatory point in RNAPIIdependent transcription. Intriguingly, recent genome-wide analyses using ChIP and whole-genome tiling arrays (ChIP–chip) in yeast, human, and Drosophila showed that the post-recruitment regulation of RNAPII-dependent transcription is much more general than previously thought [29–33]. Furthermore, the activation of transcription in mouse embryonic stem cells is generally accomplished with the release of RNAPII pausing, presumably through the recruitment of P-TEFb by the transcription factor cMyc [34]. Since germ cells begin their own transcriptional program later in development, stalling the RNAPII function by inhibiting P-TEFb may be an efficient mechanism for establishing transient transcriptional quiescence without losing transcriptional competence.
Silencing of somatic transcriptional programs also occurs in the mouse germline Unlike in Drosophila and C. elegans, the germ cells in mouse are not pre-determined by the germ plasm, but Current Opinion in Cell Biology 2010, 22:709–714
instead arise through an epigenetic mechanism in response to signals from the extraembryonic ectoderm [35,36]. PGCs in the mouse emerge as 40 alkaline phosphatase (AP)-positive and stella-positive cells that form a cluster posterior to the primitive streak in the extraembryonic mesoderm at embryonic day (E) 7.25 [6,37]. Earlier in embryogenesis, the PGC precursors reside within the proximal epiblast cells. The transcriptional regulator Blimp1 plays key roles in PGC specification [5]. Blimp1 expression is induced only in a few cells among the proximal posterior epiblast cells at E6.25–6.5, by signals from extracellular tissues [35]. These cells proliferate and contribute almost invariably to the stella-positive PGCs. In Blimp1-deficient embryos, the PGC precursors fail to migrate to and populate the embryonic gonads. Mouse PGCs arise from cells that are otherwise destined to become mesoderm, and they express a number of genes involved in mesodermal development, such as Hox, Snail, and Tbx [4,5,7]. These mesodermal genes are downregulated in Blimp1-positive PGC precursors, which in turn upregulate the expression of genes required for pluripotency (e.g. Sox2 and Nanog) as well as germ cell development (e.g. Nanos3 and Dnd1). Notably, transcriptional profiling using highly representative single-cell microarray technology indicates that Blimp1 is responsible for repressing virtually all of the transcripts that are downregulated during PGC specification [4,38]. Unlike PIE-1 in C. elegans and Pgc in Drosophila, mouse Blimp1 does not globally repress mRNA transcription during PGC specification. Nevertheless, the repression of somatic transcriptional programs is an essential process in launching the germline in mouse.
Conclusion Germline transcriptional repression has been observed in a number of animal embryos. As in Drosophila and C. elegans, PGCs in Xenopus embryos show a transient and specific repression of CTD Ser2 phosphorylation [39], although the factor responsible for the process remains unknown. Zygotic transcription in the germline appears to be repressed in ascidian embryos as well. While no downregulation of CTD phosphorylation in the germline has been detected in Halocynthia roretzi embryos [40], germline blastomeres in Ciona intestinalis embryos show a marked reduction in pSer2 signals during the cleavage stage (M.S.K., unpublished observations). Although distinct mechanisms and factors are responsible for transcriptional quiescence in different organisms, the active repression of somatic transcriptional programs during the establishment of the germline is widespread in animal development, regardless of the mode of germ cell formation.
Acknowledgements The authors thank H. Sano and M. Van Doren for comments on the manuscript. Research in our laboratory is supported in part by a Grant-inAid from the Ministry of Education, Culture, Sports and Science, Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS). www.sciencedirect.com
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