Category Archives: Placenta

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: DISCUSSION(6)

DISCUSSION(6)

Additional studies showed that an approximately 2.3-kb fragment (—6583/ —4290) contains a trophoblast-specific enhancer when inserted upstream of the SV40 basal promoter in the pGL3 promoter vector. This construct displayed enhanced lucif-erase activity in trophoblast cells relative to nontrophob-last cells, indicating that the (—6583/—4290) fragment contains trophoblast cell-specific regulatory elements. Further analysis of this approximately 2.3-kb fragment revealed that the trophoblast cell-specific elements reside in the 5′ proximal 704 bp located from 5.8 to 6.5 kb upstream of the transcription start site of the AP-2y gene. Multiple GATA transcription factor-binding sites are located in the 704-bp DNA sequence.

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: DISCUSSION(5)

The results of gene disruption studies indicate that AP-2 family members have distinct, divergent roles. The disruption of either AP-2a or AP-2$ genes showed embryonic phenotypes, with placentas largely unaffected. The AP-2a (-/-) mice displayed multiple organ malformations, including the neural tube, eye, face, forelimbs, body wall, and cardiovascular system. The AP-2fi (—/—) mice showed kidney defects. In contrast, the AP-2y gene is required for early placental function. In placental development, AP-2y has a major and unique function, and it is not required for embryonic and adult viability. The abundant expression of AP-2y in all trophoblast lineages throughout placental development, the requirement for AP-2y in the regulation of other placental genes, and the impaired placental development resulting from AP-2y gene disruption together provide strong evidence that AP-27 plays an important role in placental development. so

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: DISCUSSION(4)

DISCUSSION(4)

Combined mutation of the three sites, Sp-A, Sp-B, and Sp-C, reduced the promoter activity by more than 80%, suggesting that all three sites contribute to transcriptional activity. Indeed, these three protein-binding sites are clustered in an approximately 80-nucleotide stretch of DNA just upstream of the transcription start site. Because all these Sp sites are well conserved in human and mouse, they most likely have similar functions in the regulation of human AP-27 gene expression.

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: DISCUSSION(3)

Mutational analysis indicated that site Sp-C contributes to the transcriptional activity of the mouse AP-27 gene. Two well-conserved AP-2-binding sites are located in sites Sp-B and Sp-C of the mouse AP-27 promoter, and they overlap with the Sp sites. However, our competition assays and supershift assays with AP-2 consensus sequences and AP-2a and AP-27 antibodies failed to show that AP-2 proteins are involved in the formation of the DNA-protein complexes discussed above. there

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: DISCUSSION(2)

DISCUSSION(2)

Three Sp-binding sites (Sp-A, Sp-B, and Sp-C) were analyzed in detail by mutagenesis and electrophoretic mobility shift assays. Two protein-DNA complexes were formed with DNA corresponding to site Sp-A (—142/—123). Supershift assays indicated that one complex is composed of Sp1 and that the other complex contains Sp3. Mutational analysis showed that site Sp-A is important for the transcriptional activity of the mouse AP-27 promoter. Four pro-tein-DNA complexes are formed with DNA corresponding to site Sp-B (—107/—86). It is noteworthy that consensus Sp1 oligonucleotides competed for site Sp-B complex formation better than authentic AP-27 promoter sequence. These results suggest that the binding proteins have a higher affinity for the consensus Sp1 sequence than for the murine AP-27 promoter sequence at this site. Analysis with Sp antibodies showed that Sp1 and Sp3 are involved in these four protein-DNA complexes. Ventolin side effects

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: DISCUSSION(1)

In the present study, we cloned and characterized the promoter and 5′-flanking regions of the mouse and human AP-2y genes as an initial step in understanding the regulation of AP-2y gene expression in the trophoblast cell lineage during placental development. Primer extension and 5′ RACE experiments showed that the major transcription start site of the mouse AP-2y gene is located 241 nucleotides upstream of the ATG translation initiation codon. An atypical TATA box is located approximately 30 bp upstream of the transcription start site, suggesting that this sequence is involved in positioning the transcription initiation complex. Sequence comparison of approximately 6500 bp of 5′-flanking sequence revealed that the mouse and human AP-2y genes share little similarity except for a short, approximately 200-bp promoter proximal region that has 93% sequence identity between the human and mouse AP-2y genes.

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: RESULTS(9)

ESULTS(9)

To test the functional importance of the three Sp-binding sites, we made a series of point mutations that disrupted the three cis-elements. In Sp-A (-142/-123) site, GG was mutated to AT. In the Sp-B site (-107/-86), C was substituted with A. Three Cs were mutated to three As in the Sp-C site (-75/-50) (Fig. 9A).

The transfection studies revealed that mutational alteration of any of the three sites resulted in an approximately 20%-50% reduction in promoter activity when compared with that of the wild-type construct (-233/+181). Mutation of the Sp-A site decreased the promoter activity more than mutations at the other two sites, indicating that the Sp-A site is more important than the Sp-B and Sp-C sites. Promoter activity was decreased by 60% with combined mutations of Sp-A and Sp-B sites. Ventolin Inhaler

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: RESULTS(8)

Three DNA-protein complexes were formed using a DNA fragment (-75/-50) containing the Sp-C site and HTR-8/SVneo nuclear extracts. The nonradiolabeled wild-type probe and a probe containing the Sp1 consensus sequence diminished the abundance of the complexes, but the nonradiolabeled mutant probe and the probe with a mutant Sp1 consensus sequence showed no effect. Cold probes with both wild-type AP-2 and mutant AP-2 consensus sequences did not reduce the formation of the complexes (Fig. 8A). These data suggest that these complexes represent specific protein binding to the Sp sequence elements, but not to the AP-2 consensus sequences. Complex C3 was decreased by both wild-type and mutant Sp-C probe, but not by Sp1 and AP-2 consensus sequence probes, indicating that complex C3 resulted from interaction with nucleotide sequences other than Sp1-binding elements in the 26-bp Sp-C probe. http://ventolin-inhalers.com/

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: RESULTS(7)

RESULTS(7)

We performed a gel-supershift assay using Sp1, Sp3, and Sp4 antibodies to confirm that Sp family members bind to these sites. As shown in Figure 6B, complex A1 was supershifted by Sp1 antibody, and complex A2 was diminished by Sp3 antibody. These results indicate that complex A1 is formed from Sp1 binding, whereas complex A2 results from Sp3 binding. The Sp4 antibody did not supershift or diminish any complex. Similar results were obtained using nuclear extracts from JEG3 and Hela cells (data not shown). add comment

Sp1 and Sp3 Are Important Regulators of AP-2y Gene Transcription: RESULTS(6)

Functional Analysis of the Human AP-2gamma; Promoter and 5′-Flanking Region

We prepared five human AP-2y gene 5′ constructs. They ranged in size from approximately 200 to approximately 4300 bp and shared a common 3′ end located immediately upstream of the translation initiation codon. The 5′ end of the human AP-2-y cDNA is designated as +1. Promoter activities of the reporter genes increased as the 5′-flanking region was reduced from -4300 to -295 bp in all cell lines. Construct (-295/+166) showed the strongest promoter activity. Deletion of DNA sequence between – 295 and -130 bp abolished the promoter activity (Fig. 5), indicating that this short DNA sequence contains important cis-acting elements. buy ventolin inhaler

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