Two TFIIIA activities regulate expression of the Xenopus 5S RNA gene families.

Blanco, Jerónimo; Millstein, Larry; Razik, Mona A.; Dilworth, S. J.; Coté, Christopher; Gottesfeld, Joel M.

doi:10.1101/gad.3.10.1602

Cited by 33 publications

(21 citation statements)

References 37 publications

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“…Hardly; Xenopus TFIIIA, for example, is a well-characterized zinc finger protein unquestionably essential for transcription of the 5S rRNA gene (21) and occurring in a 7S complex with 5S rRNA (53). In stage III oocytes, TFIIIA constitutes approximately 10% of the cellular protein and is present in 10 12 molecules per cell, mostly bound to RNA (4,54). Similarly, if hnRNP K is to operate against a sink of binding sites on RNA as well as nonspecific sites on DNA, it must be an abundant protein.…”

Section: Resultsmentioning

confidence: 99%

Heterogeneous Nuclear Ribonucleoprotein K Is a Transcription Factor

Michelotti

Aronsohn

et al. 1996

Molecular and Cellular Biology

324

327

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The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Sp1 and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing singlestranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

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Section: Resultsmentioning

confidence: 99%

Heterogeneous Nuclear Ribonucleoprotein K Is a Transcription Factor

Michelotti

Aronsohn

et al. 1996

Molecular and Cellular Biology

324

327

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“…It is, however, possible that other proteins might be responsible for repressing transcription through association with the oocyte 5S RNA gene (7). We therefore carried out a number of control assays to confirm that the majority of oocyte 5S RNA genes were incorporated into nucleosomes.…”

Section: Resultsmentioning

confidence: 99%

Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo.

Chipev¹,

Wolffe²

1992

Mol. Cell. Biol.

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We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into (38). The association of histone Hi with the oocyte genes may be involved in actually establishing repression (28, 49). However, the relationship between the organization of the oocyte genes into nucleosomes, the presence or absence of histone Hi within nucleosomal DNA containing the oocyte genes, and the repressed state of the oocyte genes in vivo has not yet been resolved.In contrast to the existing analyses of the chromosomal organization of the major oocyte 5S RNA genes, no comparable study of the somatic 5S RNA genes has appeared. In this report, we have examined the chromosomal organization of both the oocyte and the somatic 5S RNA genes in erythrocytes. We find that the oocyte genes are repressed and that the whole oocyte 5S DNA repeat is organized into a positioned array of nucleosomes. Consistent with earlier work (21, 56), the internal promoter elements of the major oocyte 5S RNA gene are incorporated into a nucleosome.* Corresponding author.Disruption of this chromatin organization occurs following the removal of histone Hi. This facilitates the activation of transcription of 5S RNA genes in a complete transcription extract. Readdition of histone Hi to this active chromatin selectively represses transcription of the oocyte genes and alters the chromatin structure over the oocyte genes. In contrast, the somatic 5S DNA repeats are assembled into arrays of nucleosomes that do not appear to have any uniform organization with respect to DNA sequence. We present evidence for a region around the somatic 5S RNA gene that is accessible to nucleases and for the association of transcription factors with both the internal control region (ICR) and the 5' flanking region of this gene. MATERIALS AND METHODSPreparation of chromatin. X. laevis females were purchased from Xenopus I. Blood was collected from the heart into 1 x STM (90 mM NaCl, 10 mM Tris, 2 mM MgCI2, pH 7.4)-heparin (10 U/ml) on ice. Erythrocytes were washed twice in the same buffer by centrifugation at 600 x g for 5 min (26,56). Hepatocytes were isolated from Xenopus liver (39).Nuclei were prepared from erythrocytes and hepatocytes by established methods (23,50). Cells were Dounce homogenized, and the nuclei were resuspended in 50% glycerol-50 mM Tris HCI (pH 7.5)-100 mM KCI-5 mM MgCl2-2 mM dithiothreitol (DTT)-0.5 mM phenylmethylsulfonyl fluoride (PMSF)-0.1% ...

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“…Such compartmentalization could explain why oocyte-type class III genes are highly expressed in oocyte nuclear extracts and in microinjected oocyte nuclei (34,39) but are poorly expressed in oocyte S150 extracts. This might also explain the variation reported between different oocyte S150 extracts with respect to the relative activities for the somatic and oocyte 5S genes (5) or OAX and tMET (34; this study); depending on the method of S150 extract preparation, the contribution from the nuclear contents may vary.…”

Section: Discussionmentioning

confidence: 83%

“…The equivalent of human and yeast factor C for the Bombyx system may be represented by two essential factors, termed IIIC and IIID (35). Little information is as yet available concerning the Xenopus homolog.The differential expression of the somatic and oocyte 5S genes has been studied in detail (5,39,40,52,54). There is evidence attributing the developmental inactivation of the oocyte 5S genes to a less-effective interaction with factor C (52).…”

mentioning

confidence: 99%

Differential expression of oocyte-type class III genes with fraction TFIIIC from immature or mature oocytes.

Reynolds¹,

Johnson²

1992

Mol. Cell. Biol.

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The Xenopus OAX genes can be expressed in oocytes but are virtually inactive in somatic tissues. The tRNAMetl (tMET) genes also appear to be developmentally regulated. We have examined the reason for the differential expression of these class III genes. Analysis of the transcriptional activities of extracts derived from immature and mature oocytes revealed that the developmental regulation of these genes can be reproduced in vitro. We have partially purified the required transcription factors B and C from these extracts to ascertain the components responsible for this differential activity. The immature oocyte C fraction activates the tMET and OAX genes when reconstituted with either the immature or mature oocyte-derived B fraction. In contrast, the mature oocyte C fraction fails to activate these genes regardless of which B fraction is used. Both C fractions activated the somatic 5S gene. Purification of the oocyte C fractions by phosphocellulose or B box DNA affinity chromatography failed to separate additional activities responsible for the differential expression of OAX or tMET. By using template exclusion assays, the inability of the mature oocyte C fraction to activate transcription was correlated with an inability to form stable transcription complexes with the tMET or OAX gene.A central question in development biology concerns the mechanisms which bring about stage-specific gene expression. The Xenopus class III system provides several examples of genes which are expressed specifically in oocytes. The developmental regulation of the oocyte 5S gene has been studied in detail (for reviews, see references 16 and 36). However, the mechanisms governing the oocyte-specific expression of non-SS genes, such as OAX and tRNAMetl (tMET), are not well understood. In this study, we undertook an analysis of the factors responsible for this developmental regulation. The class III system provides a means to dissect the transcription factors responsible for stage-specific gene expression, in that the polymerase III (pol III) factors can be partially purified and recombined to reconstitute efficient transcription in vitro. At least two components, TFIIIB (factor B) and TFIIIC (factor C), are required to direct correct initiation by RNA pol III on tRNA-type class III genes (43; reviewed in 16, 36, and 45). A third factor, TFIIIA (factor A), is additionally required for transcription of 5S RNA genes (13). There is recent evidence, obtained with the Bombyx system, for an additional component of class III transcription complexes, TFIIIR, which is composed of RNA rather than protein (58).The tRNA-type promoter consists of two separable elements of -10 bp each, known as the A box and B box (16,36,45). Binding by factor C to the B box promoter element permits subsequent association by factor B, which in turn stabilizes the complex (27,32,44). For the 5S RNA gene, which lacks a B box sequence element, initial binding by factor A to the 50-bp internal promoter facilitates binding by factor C, apparently through protein-protein con...

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Two TFIIIA activities regulate expression of the Xenopus 5S RNA gene families.

Cited by 33 publications

References 37 publications

Heterogeneous Nuclear Ribonucleoprotein K Is a Transcription Factor

Heterogeneous Nuclear Ribonucleoprotein K Is a Transcription Factor

Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo.

Differential expression of oocyte-type class III genes with fraction TFIIIC from immature or mature oocytes.

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