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Identification of TSG101 functional domains and p21 loci required for TSG101-mediated p21 gene regulation.

Lin YS, Chen YJ, Cohen SN, Cheng TH - PLoS ONE (2013)

Bottom Line: A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively.Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity.Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21 (CIP1/WAF1) tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.

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Specific TSG101 domains are required for p21 promoter binding and repression.(A) Left, TSG101 contains 391 amino acids with the four indicated domains. Constructs expressing myc-tagged full-length or truncated forms of TSG101 are illustrated. Right, p21 promoter binding and repression by these TSG101 variants are summarized. ND, not determined. (B) Binding of full-length TSG101 and its truncated forms to the p21 promoter was examined by chromatin immunoprecipitation. DNA fragments, which co-precipitated with anti-myc antibody, were PCR-amplified using the primer set “d”, as shown in Figure 3A. Expression of TSG101 variants is shown by the Western blot in the bottom panel. Asterisk indicates the position of non-specific bands. (C) p21 promoter activity was assessed in Saos-2-2 cells expressing different forms of TSG101. Empty vector sample was included as control, and its promoter activity was set as 1. Data presented are the average of five independent experiments (*P<0.05, Student's t test).
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pone-0079674-g004: Specific TSG101 domains are required for p21 promoter binding and repression.(A) Left, TSG101 contains 391 amino acids with the four indicated domains. Constructs expressing myc-tagged full-length or truncated forms of TSG101 are illustrated. Right, p21 promoter binding and repression by these TSG101 variants are summarized. ND, not determined. (B) Binding of full-length TSG101 and its truncated forms to the p21 promoter was examined by chromatin immunoprecipitation. DNA fragments, which co-precipitated with anti-myc antibody, were PCR-amplified using the primer set “d”, as shown in Figure 3A. Expression of TSG101 variants is shown by the Western blot in the bottom panel. Asterisk indicates the position of non-specific bands. (C) p21 promoter activity was assessed in Saos-2-2 cells expressing different forms of TSG101. Empty vector sample was included as control, and its promoter activity was set as 1. Data presented are the average of five independent experiments (*P<0.05, Student's t test).

Mentions: Variant TSG01 transcripts lacking segments present in full-length TSG101 mRNA have been reported to accumulate in multiple types of human cancer [37]–[39]. To identify the specific TSG101 region that interacts with and regulates the p21 promoter and to establish a framework for identifying possible effects of aberrantly spliced TSG101 transcripts on p21-regulated cell proliferation, we constructed a collection of truncated variants of TSG101 (Fig. 4A, left panel). ChIP assays using these variants showed that the UEV domain alone (UEV) did not interact with the p21 promoter and that TSG101 protein lacking such domain (ΔUEV) retained the ability to bind. These results argue strongly that regulation of p21 promoter activity by TSG101 does not result from TSG101 interaction with PTAP or PSAP containing proteins, as TSG101 effects on endocytic trafficking do.


Identification of TSG101 functional domains and p21 loci required for TSG101-mediated p21 gene regulation.

Lin YS, Chen YJ, Cohen SN, Cheng TH - PLoS ONE (2013)

Specific TSG101 domains are required for p21 promoter binding and repression.(A) Left, TSG101 contains 391 amino acids with the four indicated domains. Constructs expressing myc-tagged full-length or truncated forms of TSG101 are illustrated. Right, p21 promoter binding and repression by these TSG101 variants are summarized. ND, not determined. (B) Binding of full-length TSG101 and its truncated forms to the p21 promoter was examined by chromatin immunoprecipitation. DNA fragments, which co-precipitated with anti-myc antibody, were PCR-amplified using the primer set “d”, as shown in Figure 3A. Expression of TSG101 variants is shown by the Western blot in the bottom panel. Asterisk indicates the position of non-specific bands. (C) p21 promoter activity was assessed in Saos-2-2 cells expressing different forms of TSG101. Empty vector sample was included as control, and its promoter activity was set as 1. Data presented are the average of five independent experiments (*P<0.05, Student's t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823576&req=5

pone-0079674-g004: Specific TSG101 domains are required for p21 promoter binding and repression.(A) Left, TSG101 contains 391 amino acids with the four indicated domains. Constructs expressing myc-tagged full-length or truncated forms of TSG101 are illustrated. Right, p21 promoter binding and repression by these TSG101 variants are summarized. ND, not determined. (B) Binding of full-length TSG101 and its truncated forms to the p21 promoter was examined by chromatin immunoprecipitation. DNA fragments, which co-precipitated with anti-myc antibody, were PCR-amplified using the primer set “d”, as shown in Figure 3A. Expression of TSG101 variants is shown by the Western blot in the bottom panel. Asterisk indicates the position of non-specific bands. (C) p21 promoter activity was assessed in Saos-2-2 cells expressing different forms of TSG101. Empty vector sample was included as control, and its promoter activity was set as 1. Data presented are the average of five independent experiments (*P<0.05, Student's t test).
Mentions: Variant TSG01 transcripts lacking segments present in full-length TSG101 mRNA have been reported to accumulate in multiple types of human cancer [37]–[39]. To identify the specific TSG101 region that interacts with and regulates the p21 promoter and to establish a framework for identifying possible effects of aberrantly spliced TSG101 transcripts on p21-regulated cell proliferation, we constructed a collection of truncated variants of TSG101 (Fig. 4A, left panel). ChIP assays using these variants showed that the UEV domain alone (UEV) did not interact with the p21 promoter and that TSG101 protein lacking such domain (ΔUEV) retained the ability to bind. These results argue strongly that regulation of p21 promoter activity by TSG101 does not result from TSG101 interaction with PTAP or PSAP containing proteins, as TSG101 effects on endocytic trafficking do.

Bottom Line: A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively.Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity.Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21 (CIP1/WAF1) tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.

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Related in: MedlinePlus