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Specificity protein 1 transcription factor regulates human ARTS promoter activity through multiple binding sites.

Xu F, Sun W, Li P, Chen J, Zhu D, Sun X, Wang J, Feng J, Song K, Duan Y - PLoS ONE (2015)

Bottom Line: Apoptosis-related protein in the TGF-β signaling pathway (ARTS) is an unusual mitochondrial Septin-like protein which functions as a tumor suppressor.ChIP analysis showed that Sp1 protein could bind to two of these sites (-735/-718 and -173/-157) and mutation of each Sp1 binding site led to a significant decrease in ARTS promoter activity.This would provide basis for further study on the function of ARTS on cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Biology, School of Medicine, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu, People's Republic of China; Clinical Laboratory, The Sixth People's Hospital of Nantong, Nantong 226001, Jiangsu, People's Republic of China.

ABSTRACT
Apoptosis-related protein in the TGF-β signaling pathway (ARTS) is an unusual mitochondrial Septin-like protein which functions as a tumor suppressor. There are various splice variants derived from the human Septin4 gene, one of which is ARTS, also known as Septin4_i2. Unlike other Septin4 members, ARTS can induce apoptosis in many cells, however, the underlying molecular mechanism for the transcriptional regulation of ARTS has yet to be deciphered. In this study, we attempted to analyze the promoter region of ARTS in cultured HEK-293T and LX-2 cells with the purpose of elucidating the underlying transcriptional mechanisms driving ARTS expression. We effectively demonstrated that the -824 to -5 bp region of the ARTS promoter was essential for ARTS transcription and identified four putative specificity protein 1 (Sp1) binding sites within this core promoter region. ChIP analysis showed that Sp1 protein could bind to two of these sites (-735/-718 and -173/-157) and mutation of each Sp1 binding site led to a significant decrease in ARTS promoter activity. In conclusion, all the results indicated that the Sp1 transcription factor could contribute to ARTS gene transcription. The underlying molecular events of the specific promoter of ARTS could also be used to explain why ARTS is selectively silenced during some human diseases. This would provide basis for further study on the function of ARTS on cell apoptosis.

No MeSH data available.


Related in: MedlinePlus

Sp1 binding sites are required for ARTS promoter activation.(A) The schematic representation of the mutation project. The constructs were then transiently transfected into HEK293 cells (B) and LX-2 cells (C) and promoter activities were determined by luciferase assay. The activities for the promoter of M1 and M2 plasmids were reduced in both cell lines, compared with the activity of the wild-type plasmid (*P<0.05).
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pone.0120072.g004: Sp1 binding sites are required for ARTS promoter activation.(A) The schematic representation of the mutation project. The constructs were then transiently transfected into HEK293 cells (B) and LX-2 cells (C) and promoter activities were determined by luciferase assay. The activities for the promoter of M1 and M2 plasmids were reduced in both cell lines, compared with the activity of the wild-type plasmid (*P<0.05).

Mentions: To observe the function of Sp1 in the modulation of ARTS transcription activity, site-directed mutations within the core sequence of each Sp1 site were performed, as displayed in Fig. 4A. Luciferase assays were then conducted and the luciferase activity of each mutation construct was compared with the associated wild-type construct in both LX-2 and HEK-293T cells. As evidenced in Fig. 4B and C, the luciferase activities of the Sp1 single-site mutant plasmids, M1 and M2, were significantly reduced in both cell lines compared to respective wild-type construct. These results strongly supported the hypothesis that Sp1 could positively drive ARTS gene transcription through Sp1-binding sites. Together with site-directed mutagenesis and binding analysis data, we concluded that Sp1 could contribute to the modulation of the ARTS promoter activity.


Specificity protein 1 transcription factor regulates human ARTS promoter activity through multiple binding sites.

Xu F, Sun W, Li P, Chen J, Zhu D, Sun X, Wang J, Feng J, Song K, Duan Y - PLoS ONE (2015)

Sp1 binding sites are required for ARTS promoter activation.(A) The schematic representation of the mutation project. The constructs were then transiently transfected into HEK293 cells (B) and LX-2 cells (C) and promoter activities were determined by luciferase assay. The activities for the promoter of M1 and M2 plasmids were reduced in both cell lines, compared with the activity of the wild-type plasmid (*P<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366172&req=5

pone.0120072.g004: Sp1 binding sites are required for ARTS promoter activation.(A) The schematic representation of the mutation project. The constructs were then transiently transfected into HEK293 cells (B) and LX-2 cells (C) and promoter activities were determined by luciferase assay. The activities for the promoter of M1 and M2 plasmids were reduced in both cell lines, compared with the activity of the wild-type plasmid (*P<0.05).
Mentions: To observe the function of Sp1 in the modulation of ARTS transcription activity, site-directed mutations within the core sequence of each Sp1 site were performed, as displayed in Fig. 4A. Luciferase assays were then conducted and the luciferase activity of each mutation construct was compared with the associated wild-type construct in both LX-2 and HEK-293T cells. As evidenced in Fig. 4B and C, the luciferase activities of the Sp1 single-site mutant plasmids, M1 and M2, were significantly reduced in both cell lines compared to respective wild-type construct. These results strongly supported the hypothesis that Sp1 could positively drive ARTS gene transcription through Sp1-binding sites. Together with site-directed mutagenesis and binding analysis data, we concluded that Sp1 could contribute to the modulation of the ARTS promoter activity.

Bottom Line: Apoptosis-related protein in the TGF-β signaling pathway (ARTS) is an unusual mitochondrial Septin-like protein which functions as a tumor suppressor.ChIP analysis showed that Sp1 protein could bind to two of these sites (-735/-718 and -173/-157) and mutation of each Sp1 binding site led to a significant decrease in ARTS promoter activity.This would provide basis for further study on the function of ARTS on cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Biology, School of Medicine, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu, People's Republic of China; Clinical Laboratory, The Sixth People's Hospital of Nantong, Nantong 226001, Jiangsu, People's Republic of China.

ABSTRACT
Apoptosis-related protein in the TGF-β signaling pathway (ARTS) is an unusual mitochondrial Septin-like protein which functions as a tumor suppressor. There are various splice variants derived from the human Septin4 gene, one of which is ARTS, also known as Septin4_i2. Unlike other Septin4 members, ARTS can induce apoptosis in many cells, however, the underlying molecular mechanism for the transcriptional regulation of ARTS has yet to be deciphered. In this study, we attempted to analyze the promoter region of ARTS in cultured HEK-293T and LX-2 cells with the purpose of elucidating the underlying transcriptional mechanisms driving ARTS expression. We effectively demonstrated that the -824 to -5 bp region of the ARTS promoter was essential for ARTS transcription and identified four putative specificity protein 1 (Sp1) binding sites within this core promoter region. ChIP analysis showed that Sp1 protein could bind to two of these sites (-735/-718 and -173/-157) and mutation of each Sp1 binding site led to a significant decrease in ARTS promoter activity. In conclusion, all the results indicated that the Sp1 transcription factor could contribute to ARTS gene transcription. The underlying molecular events of the specific promoter of ARTS could also be used to explain why ARTS is selectively silenced during some human diseases. This would provide basis for further study on the function of ARTS on cell apoptosis.

No MeSH data available.


Related in: MedlinePlus