Sentences with phrase «cip1 promoter»

A, DDX3 up - regulates the p21waf1 / cip1 promoter activity.

Moreover, our data also revealed that DDX3 transactivates the p21waf1 / cip1 promoter through its multiple Sp1 sites, and DDX3 collaborates with Sp1 on p21waf1 / cip1 promoter activation.

To this end, increasing amounts of expression constructs of DDX3 / DQAD, DDX3 / AAA and wild - type DDX3 were cotransfected with p21waf1 / cip1 promoter — driven luciferase reporter.

It is well known that Sp1 and Sp3 can transactivate the p21waf1 / cip1 promoter through Sp1 sites (35).

A, schematic representation of serial p21waf1 / cip1 promoter — driven luciferase reporters used in transactivation assay: p21 - Luc contains the 2.3 - kb full - length of p21waf1 / cip1 promoter (from − 2,326 to +10 nucleotide related transcription start site); its derivatives (− 159 / +8) p21 - Luc, (− 123 / +8) p21 - Luc, (− 84 / +8) p21 - Luc, (− 76 / +8) p21 - Luc, (− 63 / +8) p21 - Luc, and (− 56 / +8) p21 - Luc contain a series of deleted promoters of p21waf1 / cip1.

As shown in Fig. 2A, exogenous expression of DDX3 in various cell lines led to a 2 - to 4-fold up - regulation of p21waf1 / cip1 promoter — driven luciferase activity.

Nucleotide substitutions introduced into the regulatory elements of p21waf1 / cip1 promoter region in mutant constructs are shown in boldface and underlined.

Because DDX3 harbors growth - suppressive ability and transcriptional modulation activity of the p21waf1 / cip1 promoter, in this work we further examined the deregulated expression of DDX3 in tumors by a cancer profiling array and immunohistochemical staining analysis.

This observation is consistent with our in vitro studies (see Figs. 2 and 3), which indicate that DDX3 up - regulates p21waf1 / cip1 promoter activity in a p53 - independent manner.

As shown in Fig. 4A, transient expression of DDX3 alone resulted in a 4-fold activation of p21waf1 / cip1 promoter activity whereas transiently expressed Sp1 or Sp3 of increasing amount led to a 1.6 - to 1.8-fold activation of the p21waf1 / cip1 promoter.

The DDX3 - mediated transactivation of p21waf1 / cip1 promoter driven by mutant constructs defective in two Sp1 sites (MUT1 / 2 and MUT3 / 4), three Sp1 sites (MUT1 / 2/3 and MUT2 / 3/4), or four Sp1 sites (MUT1 / 2/3 / 4) was measured as described in (B and C).

Next, to examine the underlying mechanism by which DDX3 activates the p21waf1 / cip1 promoter, we mapped the DDX3 responsive region within the p21waf1 / cip1 promoter by serial 5 ′ - deleted promoter - driven luciferase reporters (Fig. 3A and Supplementary Fig.

The ATPase activity, but not the helicase activity, of DDX3 contributes to DDX3 - mediated transcriptional modulation on the p21waf1 / cip1 promoter.

This conclusion is further supported by another reporter assay, which was directed by a p21waf1 / cip1 promoter lacking the − 127 to − 63 nucleotide region (Fig. 3C).

Multiple Sp1 sites (Sp1 - 1, Sp1 - 2, Sp1 - 3, and Sp1 - 4) located at − 123 to − 63 related to transcription start site are essential for the transactivation of DDX3 on p21waf1 / cip1 promoter.

The mechanism of p21waf1 / cip1 induction caused by these factors often involves the action of corresponding transcription factors on the p21waf1 / cip1 promoter (17).

Interestingly, our data revealed that at a higher dosage (2 μg / well), the wild - type DDX3 and mutant DDX3 / AAA up - regulated the activity of p21waf1 / cip1 promoter ∼ 12-fold whereas the DDX3 / DQAD mutant only activated the p21waf1 / cip1 promoter activity ∼ 5-fold (Supplementary Fig.

This result strongly suggests that Sp1, but not Sp3, is involved in the transcriptional regulation of DDX3 at the p21waf1 / cip1 promoter.

Because DDX3 harbors a dominant nuclear localization in normal squamous cells and exerts a transactivation function on p21waf1 / cip1 promoter, presumably the inactivation of transcriptional modulation function of DDX3 through cytoplasmic mislocalization is relevant to the deregulation of cell growth in cancerous squamous cells.

Sp1 binds to the GC - rich sequence on the proximal region of p21waf1 / cip1 promoter and acts as a mediator for the induction of p21waf1 / cip1 gene caused by transforming growth factor β (TGF - β; ref.

Moreover, DDX3 exerted its transactivation function on p21waf1 / cip1 promoter through an ATPase - dependent but helicase - independent mechanism, and the four Sp1 sites located within the − 123 to − 63 region, relative to the transcription start site of p21waf1 / cip1 promoter, were essential for the response to DDX3.

Because DDX3 exhibits tumor suppressor functions, such as a growth - suppressive property and transcriptional activation of the p21waf1 / cip1 promoter, and is inactivated through down - regulation of gene expression or alteration of subcellular localization in tumor cells, all these features together suggest that DDX3 might be a candidate tumor suppressor.

(− 2,326 / +8) Δ (− 127 / − 63) p21 - Luc is also a derivative of p21 - Luc reporter containing the full - length p21waf1 / cip1 promoter lacking − 127 to − 63 region.

p21waf1 / cip1 promoter — driven luciferase reporter (p21 - Luc; 0.05 - 0.5 μg) and increasing amount (0.1 - 2 μg) of FLAG - DDX3 expression construct were cotransfected into various cell lines as indicated.

Interestingly, the induction effect of DDX3 on the p21waf1 / cip1 promoter was increased to 10-fold when coexpressing with Sp1; however, this coactivation effect was not found when coexpressing with Sp3 (Fig. 4A).

To examine whether DDX3 transactivates the p21waf1 / cip1 promoter activity through Sp1 site - interacting proteins, the Sp1 or Sp3 expression plasmid together with the DDX3 expression construct and the p21waf1 / cip1 promoter — driven luciferase reporter were introduced into HuH - 7 cells.

B and C, HuH - 7 cells were cotransfected with a p21waf1 / cip1 promoter — driven reporter (p21 - Luc) or its derivatives represented in (A)(0.25 μg of each) together with DDX3 expression construct (pcDNA3 - SRα / FLAG - DDX 3) or control vector (pcDNA3 - SRα / FLAG; 2 μg).

The fold transactivation of the various p21waf1 / cip1 promoter — directed reporter constructs by DDX3 is shown as fold activation relative to the control transfection and is derived from at lease three independent experiments done in triplicate.

The fold transactivation of the p21waf1 / cip1 promoter — driven reporter elicited by DDX3 alone or together with Sp1 or Sp3 is presented as fold activation relative to the control transfection and is derived from at lease three independent experiments done in triplicate.

The p21waf1 / cip1 promoter — driven luciferase reporters (pGL3 derivatives) containing point mutations in six Sp1 elements, (Mut1) p21 - Luc, (Mut2) p21 - Luc, (Mut3) p21 - Luc, (Mut4) p21 - Luc, and (Mut5 / 6) p21 - Luc and the parental wild - type reporter, (Wt) p21 - Luc, were kindly provided by Dr. D. Kardassis (Department of Basic Sciences, University of Crete Medical School, Heraklion, Crete, Greece; ref.

17) is comparable with the full - length p21waf1 / cip1 promoter — driven reporter, p21 - Luc (see Supplementary Fig.

Most interestingly, our data also reveal that, like other p21waf1 / cip1 promoter — regulating factors acting on this GC - rich responsive region, such as Smad3 (24), KLF6 (37), and c - Myc (38), DDX3 up - regulates p21waf1 / cip1 promoter through physical interaction with Sp1 (see Fig. 4).

A, functional interaction between DDX3 and Sp1 on p21waf1 / cip1 promoter.

Not exact matches

DDX3 enhances p21waf1 / cip1 gene expression through up - regulation of promoter activity of p21waf1 / cip1.

DDX3 enhances the expression of p21waf1 / cip1 gene and up - regulates the promoter activity of p21waf1 / cip1 through an ATPase - dependent but helicase - independent mechanism.

To construct the p21waf1 / cip1 internal promoter region (nucleotide − 127 to − 63) deleted reporter plasmid (− 2,325 / +8) Δ (− 127 / − 63) p21 - Luc, the p21 - Luc construct was digested with ApaI, blunted, and then self - ligated.

Moreover, in this work, the DDX3 - responsive region is delineated to 60 bp (between − 123 and − 63) of the p21waf1 / cip1 proximal promoter region (see Fig. 3B), and the integrity of multiple tandem Sp1 sites in this region is required for the activation induced by DDX3 (see Fig. 3C).

Furthermore, DDX3 interacted and cooperated with Sp1 to up - regulate the promoter activity of p21waf1 / cip1.

To address the mechanism of growth inhibition by DDX3, we examined whether the promoter activity and the expression level of the cell cycle regulator p21waf1 / cip1 are regulated by DDX3.