Yin Yang 1 regulates the transcriptional repression of Survivin

Abstract

The mechanisms for regulation of the Inhibitor of Apoptosis (IAP) Survivin in cells undergoing stress associated with tumor development and the tumor microenvironment are not well understood. The stress response transcription factors HIF-1α and Yin Yang 1 (YY1) were hypothesized to contribute to the upregulation of Survivin in tumor cells. As expected, U2OS cells overexpressing HIF-1α showed a 2- to 3-fold transactivation when transfected. Surprisingly, when YY1 was overexpressed in this survivin promoter reporter system, luciferase expression was repressed 30- to 40-fold. YY1 involvement in survivin promoter repression was confirmed using siRNA directed against YY1. These studies showed that knockdown of YY1 releases the survivin promoter from the observed repression and leads to a 3- to 5-fold increase in promoter activity above basal levels. A U2OS cell line containing a stable YY1 Tet-off system was used to determine whether a temporal increase in YY1 expression affects Survivin protein levels. A low to moderate decrease in Survivin protein was observed 24h and 48h after Tet removal. Studies also confirmed that YY1 is capable of directly binding to the survivin promoter. Collectively, these findings identify novel basal transcriptional requirements of survivin gene expression which are likely to play important roles in the development of cancer and resistance to its treatment.

Keywords: HIF-1; IAP; Survivin; Transcriptional repressor; YY1.

Fig. 1

Effect of HIF-1, and YY1 overexpression on survivin promoter activity and transcript levels. (A) Proximal survivin promoter schematic. Analysis revealed multiple putative YY1 binding sites (bolding), HIF-1 binding sites (boxed segments), and heat shock elements (GAA). For reference, putative SP1 sites are denoted as underlined segments. (B) Luciferase reporter assays were performed using survivin promoter constructs ranging in length from +6280 bp to +230 bp. Error bars represent the standard deviation of duplicate luminescence measurement. Results are representative of two repeat experiments. (C) RT-PCR analysis of survivin expression following overexpression of YY1, and HIF-1. Cells were transfected with the corresponding empty vector for each transcription factor. (D) YY1 siRNA relieves the survivin promoter from transcriptional repression. Luciferase reporter assays were performed after YY1 overexpression and siRNA knockdown in (D) U2OS and (E) Panc-1 cells. Three survivin promoter reporter constructs were tested (pluc1430, pLuc 393, and pLuc 281). Relative expression indicates promoter activity relative to luciferase activity in the presence of empty vector and background activity. Error bars represent the standard deviation of duplicate luminescence measurements. Results are representative of two repeated experiments.

Fig. 2

Survivin expression decreases after 48 h of YY1 overexpression. Western blot analysis of Survivin expression after YY1 overexpression via tet-off U2OS cells was analyzed. (A) U2OS cells that stably express a YY1 overexpression vector under the control of a tetracycline responsive promoter were lysed and protein was extracted for western blot analysis. +/− indicates the presence or absence, respectively, of tet in the culture media. Data is representative of 3 independent experiments. (B) Densitometric analysis of Western blot bands. Bars represent density of YY1 or Survivin normalized to the actin band density.

Fig. 3

Mutation of two putative YY1 binding sites in the proximal survivin promoter alters promoter activity (A) The two putative YY1 binding sites (contained within pLuc230 construct) were mutated from the core YY1 recognition site CAT to GGG. (B) Luciferase reporter assay. U2OS cells were transfected with either (1) pLuc230, the standard pGL3 vector containing 230 unmutated bp of the survivin promoter, or (2) pLucMut in which the two putative YY1 binding sites were mutated from CAT to GGG. Each vector was cotransfected with either empty pcDNA, YY1, or YY1 siRNA as well as pRL-tk for transfection efficiency internal control. Error bars represent standard deviation of duplicate luminescence measurement, and results are representative of multiple experiments.

Fig. 4

YY1 directly interacts with the survivin promoter. (A) Schematic of survivin promoter representing regions investigated for YY1 binding. (B) Electrophoretic mobility shift assay. Nuclear extract was prepared from U2OS cells. 32p labelled probes were incubated with nuclear extracts either alone (Lanes 1, 4, 7 and 10), with anti-YY1 antibody (lanes 2, 5, 8 and 11) or cold competitor probes in 100× excess (lanes 3, 6, 9 and 12). Arrow indicates YY1 bound to probe. * indicates supershift. (C) Comparison of human and mouse survivin promoter sequences. Boxed segments represent the 2 putative YY1 binding segments of the survivin promoter contained within the pLuc230 construct that were mutated in previous experiments. There is lack of homology between mouse and human at both putative YY1 binding sites found in the first 230 bp of the survivin promoter.