Activation Functions of Transcription Factor Sp1 at U2 snRNA and TATA Box Promoters

Forsberg, Maud; Ström, Anne-Christine; Lillhager, Peter; Westin, Gunnar

doi:10.1515/bchm3.1995.376.11.661

Cited by 9 publications

(10 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Comparison of the four repeats revealed the presence of seven invariant residues located at positions 1,5,7,8,11,12, and 15 in each repeat (Fig. 7A).…”

Section: Resultsmentioning

confidence: 99%

“…Based on our work, we propose that the two separate Staf activation domains are involved in two different transactivation mechanisms: one for the specific activation of mRNA promoters and the other for that of snRNA and snRNA-type promoters. Forsberg et al (11) showed that Sp1 is able to stimulate transcription from mRNA and snRNA promoters. However, it must be pointed out that in this case overlapping domains are involved, while we identified two distinct, specialized domains in Staf.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Two Distinct Domains in Staf To Selectively Activate Small Nuclear RNA-Type and mRNA Promoters

Schuster

Krol

Carbon

1998

Molecular and Cellular Biology

View full text Add to dashboard Cite

Staf is a transcriptional activator of prime importance for enhanced transcription of small nuclear (snRNA) and snRNA-type genes transcribed by RNA polymerases II and III (Pol II and III). In addition to this activity, it also possesses the capacity to stimulate expression from an RNA polymerase II mRNA promoter. This promiscuous activator thus provides a useful model system for studying the mechanism by which one single transcription factor can activate a large variety of promoters. Here, we report the use of in vivo assays to identify the Staf activation domains involved in promoter selectivity. Analysis of Staf mutants reveals the existence of two physically and functionally distinct regions, outside of the DNA binding domain, responsible for mediating selective transcriptional activation. While a 93-amino-acid domain, with the striking presence of four repeated units, is specialized for transcriptional activation of an mRNA promoter, a segment of only 18 amino acids, with a critical Leu-213 residue, acts specifically on Pol II and Pol III snRNA and snRNA-type promoters. In addition, this study disclosed the fundamental importance of invariant leucine and aspartic acid residues located in each repeat unit of the mRNA activation domain. Staf is therefore the first transcriptional activator described so far to harbor two physically and functionally distinct activator domains. This finding suggests that the same activator can contact different, specialized transcription complexes formed on different types of basal promoters through promoter-specific transactivation pathways.Transcriptional activation in eukaryotes is achieved by regulatory proteins recruiting the transcriptional machinery and chromatin remodeling factors to the promoter. Transcriptional activators have been shown to present a modular organization consisting of distinct domains for sequence-specific DNA binding and transcriptional activation through interaction with other proteins (for a review, see reference 34). DNA binding domains of many transactivators fall into readily identifiable classes. Instead, domains responsible for transcriptional activity may not possess such a well-defined organization. While no obvious sequence similarity has been detected among the activation domains of diverse activator proteins, they nevertheless often carry a distinctive amino acid composition. For example, activation domains particularly rich either in prolines, glutamines, acidic amino acids, or hydroxylated amino acids have been described (for reviews, see references 4, 21, 27, and 51). Despite the identification of targets for numerous activation domains and elucidation of the functional importance of particular residues, the structural basis for the capacity of an activation domain to stimulate transcription remains poorly understood. However, recent structural studies suggest that the ␣-helix may be a common structural motif in the binding of transactivation factors to transcriptional activation factors (22,52). Transcriptional activators were also desc...

show abstract

“…Comparison of the four repeats revealed the presence of seven invariant residues located at positions 1,5,7,8,11,12, and 15 in each repeat (Fig. 7A).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Two Distinct Domains in Staf To Selectively Activate Small Nuclear RNA-Type and mRNA Promoters

Schuster

Krol

Carbon

1998

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…The Gal4-Sp1 fusion protein contained the first 612 amino acids of the Sp1 protein and a Gal4-DBD at the C-terminus [48]. The Sp1-specific sequence within this fusion protein includes the major activation domains A, B and most of C, but lacks the activation domain D and the DBD [49].…”

Section: Pma Does Not Increase Expression or Transcriptional Activitymentioning

confidence: 99%

“…(B) Effect of Gal4-Sp1 or Gal4-DBD on the wild-type or Gal4-P3 promoter in the presence or absence of PMA. The Gal4-Sp1 fusion protein contains the first 612 amino acids of the Sp1 protein, whereas the Sp1-DBD at the C-terminus was replaced by the Gal4-DBD [48]. The Gal4-PTHrP P3 promoter contains a Gal4-binding site between − 63 and − 47, which replaces the Sp1-binding site (Figure 2A) [21].…”

Section: Pma Abrogates Binding Of An Mcf-7 Nuclear Protein To a Sequementioning

confidence: 99%

Ets2 and protein kinase Cepsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells

Lindemann

Braig

Hauser

et al. 2003

Biochemical Journal

View full text Add to dashboard Cite

Parathyroid hormone-related protein (PTHrP) promotes the metastatic potential and proliferation of breast cancer cells, and acts anti-apoptotically. In invasive MDA-MB-231 breast cancer cells, transforming growth factor β-regulated PTHrP synthesis is mediated by an Ets1/Smad3-dependent activation of the PTHrP P3 promoter. In the present study, we studied the regulation of PTHrP expression in non-invasive, Ets1-deficient and transforming growth factor β-resistant MCF-7 cells. We found PMA to be a strong stimulator of P3-dependent PTHrP expression in MCF-7 cells. Mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase 1 (MEK-1)/ERK1/2 inhibitor PD98059 interfered with this activity. Promoter studies revealed that the PMA effect depended on the Ets and stimulating protein-1 (Sp1)-binding sites. Of several Ets factors tested, Ets2, but not Ese-1, Elf-1 or Ets1, supported the PMA-dependent increase in promoter activity. PD98059 and a threonine to alanine mutation of the ERK1/2-responsive Ets2 phosphorylation site at position 72 inhibited the Ets2/PMA effect. Activated protein kinase C (PKC)ε could mimic PMA by stimulating the P3 promoter alone or in co-operation with Ets2 in an MEK-1/ERK1/2-dependent manner. Activated PKCα, although capable of co-operating with Ets2, failed to induce transcription from the P3 promoter on its own. The Ets2/PKCα synergistic effect was neither sensitive to PD98059 nor to Thr 72 /Ala 72 mutation. PMA neither increased the expression of Sp1 nor modulated the transcriptional activity of Sp1. However, it induced the displacement of a yet unknown factor from the Sp1-binding site, which may result in Sp1 recruitment to the promoter. Our results suggest an ERK1/2-dependent Ets2/PKCε synergism to be involved in PTHrP expression in MCF-7 breast cancer cells.

show abstract

“…The plasmid -120/TATA(GAA) was constructed by changing the sequence (-30)TAGAAA(-25) to (-30)GAAAAA(-25), and the plasmid-120/TATA(G3T) was constructed by substituting the sequence ( -30)TAG-AAA(-25) for (-30)TATAAA(-25). To construct a reporter gene with GAL4 binding sites immediately upstream of the minimal LRP-2 promoter (-35 to +52), an oligonucleotide with Kpnl overhangs containing two tandem copies of the UAS (Forsberg et al, 1995) was ligated to Ä^nl-digested -35/wt. All constructs were verified by sequencing.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Characterization of the HumanMegalin/LRP-2PromoterIn Vitroand in Primary Parathyroid Cells

Knutson

Hellman²,

Åkerström³

et al. 1998

DNA and Cell Biology

Self Cite

View full text Add to dashboard Cite

The gp330/Megalin/LRP-2 protein belongs to the low-density lipoprotein receptor gene family and is believed to function as an endocytic receptor for the uptake of lipoproteins and many other ligands. Other functions of this protein may include a role in calcium sensing in the parathyroid glands and other tissues. In order to study the transcriptional regulation of the human LRP-2 gene, a clone containing the 5'-flanking region was isolated from a genomic DNA library, and a transient transfection protocol for primary bovine parathyroid cells was established. RNA mapping techniques located the transcriptional start site 136 bp upstream of the initiation codon. Transient expression in several cell types, including primary parathyroid cells, and in vitro transcription in HeLa cell nuclear extracts showed that sequences between -120 and -35 were important for activated transcription. This region contains consensus binding sites (GC boxes) for transcription factor Sp1. Mutation of the GC boxes abolished binding of Sp1 in vitro and resulted in reduced transcription in vitro and in transfected cells. Furthermore, Sp1 stimulated transcription when tethered to the LRP-2 core promoter through a heterologous DNA-binding domain. Through site-directed mutagenesis, we identified a novel atypical TATA element with the sequence TAGAAAA. Intriguingly, this sequence motif was shown previously not to mediate transcription in a systematic mutational analysis of the TATA motif. Possible roles of this novel TATA element in the regulation of transcription initiation are discussed. The isolation and characterization of the LRP-2 promoter and the 5'-flanking region and the establishment of a transient expression assay in primary parathyroid cells will facilitate studies on the regulatory mechanisms of the LRP-2 gene and of other genes expressed in the parathyroid glands.

show abstract

Activation Functions of Transcription Factor Sp1 at U2 snRNA and TATA Box Promoters

Cited by 9 publications

References 32 publications

Two Distinct Domains in Staf To Selectively Activate Small Nuclear RNA-Type and mRNA Promoters

Two Distinct Domains in Staf To Selectively Activate Small Nuclear RNA-Type and mRNA Promoters

Ets2 and protein kinase Cepsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells

Characterization of the HumanMegalin/LRP-2PromoterIn Vitroand in Primary Parathyroid Cells

Contact Info

Product

Resources

About