Restricted Specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes

Abstract

Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA. The wild-type factor and the alanine variant activate transcription of somatic-and oocyte-type 5S rRNA genes in nuclear extract immunodepleted of endogenous TFIIIA. The glutamic acid variant (S16E) supports the transcription of somatic-type genes at levels comparable to those of wild-type TFIIIA; however, there is no transcription of the oocyte-type genes. This differential behavior of the phosphomimetic mutant protein is also observed in vivo when using early-stage embryos, where this mutant failed to activate transcription of the endogenous oocyte-type genes. Template exclusion assays establish that the S16E mutant binds to the oocyte-type 5S rRNA genes and recruits at least one other polymerase III transcription factor into an inactive complex. Phosphorylation of TFIIIA by CK2 may allow the factor to continue to act as a positive activator of the somatic-type genes and simultaneously as a repressor of the oocyte-type 5S rRNA genes, indicating that there is a mechanism that actively promotes repression of the oocyte-type genes at the end of oogenesis.The synthesis of 5S rRNA during Xenopus oogenesis and embryogenesis provides a paradigm for developmental control of transcription (79). The oocyte-type 5S rRNA genes, which number more than 20,000 per haploid genome, are transcribed during oogenesis and briefly during early embryogenesis. The 400 somatic-type genes are active at all stages of development. Formation of transcription initiation complexes on the internal promoters of the 5S rRNA genes requires the initial binding of transcription factor IIIA (TFIIIA), followed by the ordered addition of TFIIIC and TFIIIB (46). Despite minor differences in the sequences of the two types of 5S rRNA genes, TFIIIA binds to the internal promoters of both with equal affinity (52). TFIIIC, however, preferentially binds to and stabilizes the complex of TFIIIA on the somatic-type genes (44, 79). Thus, the differential transcription of the two 5S rRNA genes during early development could, at least in part, be due to the levels of transcription factors. Nonetheless, the principal mediator of 5S rRNA gene transcription from gastrulation onward is chromatin structure.Histone H1 orchestrates the repression of oocyte-type genes in somatic cells (5,21,66,78). This inhibition occurs after the midblastula transition (MBT), when adult H1A begins to replace the maternal histone H1 variant, H1M (20,21,40). Nucleosomes are arranged differently over the two types of 5S RNA genes in somatic cells (10, 30, 83) because histone H1 acts as an architectural determinant (58, 67). In the presence of the linker histone, a stable nucleosome is positioned on the oocyte 5S rRNA gene that prevents binding of TFIIIA,...

DNA-methylation dependent regulation of embryo-specific 5S ribosomal DNA cluster transcription in adult tissues of sea urchin Paracentrotus lividus

“…Indeed, in a recent paper describing a study 165 of the sea urchin P. lividus, we demonstrated not only that all clusters are 166 transcribed, but also that the 700 bp cluster exhibited a high level of 167 heterogeneity in terms of sequence [25]. We also demonstrated that a 168 regulation exists during the developmental stage and in adult tissue, 169 as reported in Xenopus laevis [31][32][33][34]. In this organism, in particular, ants are substituted by somatic-specific ones.…”

Small Ubiquitin-like Modifier (SUMO)-mediated Repression of the Xenopus Oocyte 5 S rRNA Genes

“…Consistent with this position of the amino acid, we found no evidence that phosphorylation of TFIIIA affects its binding affinity for the promoters of either the oocyte-or somatic-type genes nor for 5 S rRNA, suggesting that the functional effect of modification likely alters an interaction with another protein (24). To address this question, we carried out a yeast two-hybrid screen of a Xenopus expression library using the first four fingers of TFIIIA as bait and identified PIAS2b, a SUMO ligase, and XCtBP, a transcriptional corepressor regulated by SUMOylation, as candidates.…”

“…Substitution with glutamic acid at this position serves as a putative phosphomimetic that cannot support transcription of the oocyte-type genes in vitro or in vivo despite binding and recruiting at least one other polymerase III transcription factor (24). This observation suggests that this region of TFIIIA is involved in an interaction that regulates the differential transcription of 5 S rRNA genes.…”

“…Phosphorylation begins during oogenesis and appears to be stimulated by progesterone-induced maturation of the oocyte to an egg (24). TFIIIA, in which serine 16 is replaced with glutamic acid, cannot support the transcription of oocyte-type 5 S rRNA genes in vitro or in vivo, whereas transcription of the somatic genes is unaffected.…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part II. Kinase Regulators and Substrates

“…The incorporation of histone H1 into chromatin during Xenopus embryogenesis directs the specific repression of the TFIIIA-activated transcription of 5S rRNA genes (Bouvet et al 1994). Phospho-form of TFIIIA may allow the factor to act as repressor for oocyte-type 5S rRNA genes (Ghose et al 2004). TFIIIA favorably binds to the somatic nucleosome whereas H1 preferentially binds to the oocyte nucleosome, excluding TFIIIA binding in Xenopus oocyte (Panetta et al 1998).…”