XBP1 negatively regulates CENPF expression via recruiting ATF6α to the promoter during ER stress

Abstract

Background: Centromere protein F (CENPF) is a key component of the kinetochore complex involved in mitosis, cell differentiation and cellular response to stresses. However, the alteration of CENPF in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we investigate CENPF regulation in response to ER stress.

Methods: Quantitative real-time polymerase chain reaction and western blotting were used to determine CENPF expression under ER stress. Luciferase activity analysis was performed to investigate the promoter regions contributing to CENPF transcription in response to TG. Chromatin immunoprecipitation (ChIP) and ChIP Re-IP assays were used to determine if X-box binding protein 1 (XBP1) and/or activating transcription factor 6α (ATF6α) bind in the CENPF promoter region. Cell apoptosis and proliferation were analyzed using TUNEL, cell growth and clonogenic assays.

Results: CENPF expression is dramatically reduced under ER stress induced by thapsigargin (TG), brefeldin A (BFA), or tunicamycin (TM) and this downregulation of CENPF expression was dependent on XBP1 and ATF6α. Luciferase activity analysis of the truncated CENPF promoter indicates that regions from bases - 679 to - 488 and from - 241 to - 78 in the CENPF promoter were sensitive to TG treatment. Additionally, ChIP and ChIP Re-IP assays reveal that XBP1 and ATF6α were assembled on the same regions of CENPF promoter. Notably, we identify two XBP1 binding sequences at positions - 567 and - 192, to which XBP1 binding was enhanced by TG. Finally, CENPF overexpression inhibits cell apoptosis and promotes cell proliferation in response to ER stress.

Conclusion: In summary, these results demonstrate that ER stress plays a crucial role in CENPF expression, and XBP1 may up-regulate DNA-binding affinities after TG treatment to the promoter of CENPF. These findings may contribute to the understanding of the molecular mechanism of CENPF regulation.

Keywords: ATF6α; CENPF; ER stress; Expression regulation; XBP1.

Fig. 1

CENPF expression is downregulated in osteosarcoma U2OS cells in response to ER stress. a U2OS cells were treated with DMSO vehicle (control), BFA (1 μg/ml), TG (1 μM) or TM (2.5 μg/ml) for 0, 6, or 24 h. The CENPF mRNA levels were quantified by real-time reverse transcription-PCR (q-RT-PCR) and normalized to 18S RNA. b Changes in CENPF protein levels induced by BFA, TG and TM treatment in U2OS cells. CENPF and α-tubulin levels were determined by western blot analysis. Data are representative of 3 independent experiments. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 2

ER stress-induced decrease in CENPF expression is dependent on ATF6 and XBP1. a q-RT-PCR analysis of CENPF mRNA level in U2OS cells with or without transient knockdown of IRE1α, PERK and ATF6. b Western blotting analysis of CENPF, IRE1α, PERK and ATF6 protein level in U2OS cells with or without transient knockdown of IRE1α, PERK and ATF6. c q-RT-PCR analysis of CENPF mRNA level in U2OS cells with or without transient knockdown CHOP, XBP1 and ATF4. d Western blotting analysis of CENPF protein level in U2OS cells with or without transient knockdown of CHOP, XBP1 and ATF4. e 1 μg XBP1 spliced expression vector was transiently transfected into U2OS cells as indicated, and the mRNA level was quantified by q-RT-PCR analysis. f Different dose of XBP1 spliced expression vector was transiently transfected into U2OS cells as indicated, and CENPF protein level was estimated by western blot analysis. Data are representative of 3 independent experiments. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 3

Transient transfection analysis of CENPF gene promoter constructs. a Using TRANSFAC-TESS and Match™ on-line softwares, we identified potential transcription factor binding motifs in the CENPF promoter. b Schematic representation of a series of 5′-deletion CENPF promoter luciferase constructs. Numbering is defined relative to the transcription start site. c Construction of a series of 5′-truncated CENPF promoters and their relative luciferase activities without (the white bar) or with (the black bar) 24 h TG treatment. Luciferase activities were determined and normalized to Renilla activity. Results are expressed as a percentage of the untreated control, which is taken as 100%. Data are representative of 3 independent experiments. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 4

ChIP and ChIP Re-IP assays analysis of TG-induced binding of XBP1 and ATF6 to the CENPF promoter in vivo. a ChIP assay to examine binding of XBP1 to the CENPF promoter region (− 679 to − 488) by TG in vivo. b ChIP assay to examine binding of XBP1 to the CENPF promoter (− 241 to − 78) by TG in vivo. c ChIP Re-IP to examine whether XBP1and ATF6 were assembled on the same CENPF promoter region (− 679 to − 488). d ChIP Re-IP to examine whether XBP1and ATF6 were assembled on CENPF promoter region (− 241 to − 78). Data are representative of 3 independent experiments. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 5

Mutagenesis analysis of XBP1 binding motifs involved in TG-induced transcriptional activity of the CENPF promoter. a Construction of CENPF promoter/luc vectors with XBP1 binding elements. Wild-type, mutant and deletion XBP1 binding elements (position − 567) are denoted as p680-luc, p680m-luc and p680d-luc, respectively. Wild-type, mutant and deletion XBP1 binding element (position − 192) are denoted by p242-luc, p242m-luc and p242d-luc, respectively. b, c Relative luciferase activities with or without TG treatment for 24 h. Measured luciferase activities were normalized to Renilla luciferase activities. Results are expressed as a percentage of the untreated control that is taken as 100%. Data are representative of 3 independent experiments. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 6

Overexpression of CENPF inhibits cell apoptosis and promotes cell proliferation in response to ER stress. a U2OS cells transfected with or without transient overexpression of CENPF were incubated with 1 μM TG for 24 h, and apoptotic cells were counted using a TUNEL assay. b U2OS cells transfected with or without transient overexpression of CENPF were incubated with or without TG for 24 h. Viable cells were counted using trypan blue exclusion. c U2OS cells transfected with control or CENPF plasmid were incubated with or without TG for 7 days. Colonies were stained with crystal violet and counted. Data are representative of 3 independent experiments. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001