Different Potential of Cellular and Viral Activators of Transcription Revealed in Oocytes and Early Embryos of<i>Xenopus laevis</i>

Xu, Licen; Rungger, Duri; Georgiev, Oleg; Seipel, Katja; Schaffner, Walter

doi:10.1515/bchm3.1994.375.2.105

Cited by 16 publications

(16 citation statements)

References 24 publications

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“…In our experiment, where the excess of GAL4 over GAL4 binding sites is only 4, we observe a premature transcription only if GAL4-VP1 6 is pre-bound to DNA before injection ( Figure 6B). Transcription is not detected before MBT if GAL4-VP16 is not pre-bound to the DNA template ( Figure 3B and independent recent result of Xu et al, 1994). These data are consistent with our conclusion that a dynamic competition between chromatin assembly and the assembly of transcription complexes regulates gene expression during early development (Prioleau et al, 1994).…”

Section: Resultssupporting

confidence: 91%

“…Figure 3B shows that transcription remains repressed during early development, despite the addition of GAL4-VP16. A similar observation was recently reported by Xu et al (1994). However, when MBT is reached, the developmental reactivation of transcription occurs, which is entirely dependent on the presence of GAL4-VP16 protein ( Figure 3B).…”

Section: Resultssupporting

confidence: 89%

“…Cis-acting proteins can have access to their sites in this repressive context, and a footprint is readily detected (Figure 4). However, the recruitment of the basal machinery cannot occur ( Figure 2B and Xu et al, 1994). After the MBT, most of the histones in excess have been titrated, and a somaticlike histone/DNA ratio is reached.…”

Section: I)cmentioning

confidence: 99%

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Programming of a repressed but committed chromatin structure during early development.

1995

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The determination of chromatin for transcription during early development as well as the requirement for trans‐acting factors during this period has been analysed in Xenopus. Basal transcription is repressed both during oogenesis and after the mid‐blastula transition (MBT), and transactivators are required to relieve this repression. In contrast, transactivators cannot overcome the generalized transcriptional repression which occurs in embryos before MBT. However, they do bind to promoters leading to a repressed but preset chromatin structure. Experiments involving the pre‐binding of TATA binding protein (TBP) or of the strong transactivator GAL4‐VP16 further show that there is no limiting factor before the MBT, and that it is the recruitment and stabilization of the basal transcription machinery and not of transactivators which is repressed during early development. This multi‐step process in gene activation, with activation of promoters temporally uncoupled from their commitment, may be of importance in the regulation of early embryonic events by providing molecular signposts for future determinations.

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 89%

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Programming of a repressed but committed chromatin structure during early development.

1995

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“…An RNA amount equivalent to two embryos was used. S1 mapping was performed using an oligonucleotide probe as described previously (Westin et al ., 1987; Xu et al ., 1994).…”

Section: Methodsmentioning

confidence: 99%

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA

et al. 1998

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In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF-κB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by α-amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replicationdependent, possibly passive mechanism.

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“…We also tested the GAL4-(DDFDL) 4x construct in Xenopus oocytes. It activated transcription very strongly, showing that the activation function of this simple module was not restricted to mammalian cell lines (Seipel, unpub-lished; see also Xu et a/., 1994). In order to test the importance of the phenylalanine residue within the five amino acid sequence we designed constructs with multiple copies of the sequence DDLDL.…”

Section: Minimal Sequence Requirement For Transcriptional Activationmentioning

confidence: 99%

A Minimal Transcription Activation Domain Consisting of a Specific Array of Aspartic Acid and Leucine Residues

Seipel¹,

Georgiev²,

Schaffner³

1994

Biological Chemistry Hoppe-Seyler

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Transcriptional activation by the herpesvirus protein VP16 (= Vmw65, aTlF) is mediated by its C-terminal acidic activation domain. Using GAL4 fusion proteins, we have previously shown that a construct containing two tandem copies of a short eleven amino acid fragment derived from the VP16 domain (DALDDFDLDML, residues 437-447) activates transcription in mammalian cells with an efficiency comparable to a GAL4 fusion with the full VP16 activation domain (residues 413-490). Here we have mutagenized this eleven amino acid core sequence and find that a mutant sequence with little inherent activity can cooperate with a wildtype sequence to yield almost full activity. Moreover, greater activity is observed when the wildtype sequence is positioned at the distal, rather than the proximal, end of the fusion protein, indicating that the distal position facilitates contacts to the transcription apparatus. We have also further reduced the eleven amino acid activating sequence to shorter sequence motifs. Two copies of eight and seven amino acids (DALDDFDL and DDFDLDL, respectively), or four copies of the sequence motif DDFDL are required to reach the activation potential of two eleven amino acid motifs. Four copies of the sequence DDLDL still activate transcription strongly (up to two-thirds of DDFDL), indicating that an aromatic residue is not an essential feature of this type of activation domain. However, repetitions of DDL or DL do not yield activity. Thus the minimal requirement for transcriptional activation is the presence of a sequence of some fifteen to twenty amino acids consisting of a specific array of aspartic acid and leucine residues. The motif DDLDL could be a prototypic activation module of the acidic/ hydrophobic class of activation domains.

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Different Potential of Cellular and Viral Activators of Transcription Revealed in Oocytes and Early Embryos ofXenopus laevis

Cited by 16 publications

References 24 publications

Programming of a repressed but committed chromatin structure during early development.

Programming of a repressed but committed chromatin structure during early development.

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA

A Minimal Transcription Activation Domain Consisting of a Specific Array of Aspartic Acid and Leucine Residues

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