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LincRNA-p21 acts as a mediator of ING1b-induced apoptosis.

Tran UM, Rajarajacholan U, Soh J, Kim TS, Thalappilly S, Sensen CW, Riabowol K - Cell Death Dis (2015)

Bottom Line: We found that this function of lincRNA-p21 is conserved in human cell models.However, their effects become more additive under conditions of stress.Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Oncology, University of Calgary, 3330 Hospital Drive NW, Calgary, T2N 4N1 Alberta, Canada.

ABSTRACT
ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions.

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ING1b requires the NLS, UBD/PBR and LID domains to control lincRNA-p21 expression. (a) A comparison of ING1 domains and a representation of the ING1b deletion constructs that were used in this study. (b, c and d) Hs68 fibroblasts were treated with either an empty control vector or a deletion mutant of ING1b lacking the plant homeo domain (PHD), nuclear localization signal (NLS), ubiquitin-binding domain/polybasic region (UBD/PBR) or lamin interaction domain (LID) for 48 h before collection of RNA and quantification of lincRNA-p21 expression by qRT-PCR. RNA levels were normalized to GAPDH expression and compared with controls. ING1b, p53 and α-tubulin protein expression levels were measured using western blot analysis. All experiments were carried out in triplicate and asterisks indicate P<0.05)
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fig5: ING1b requires the NLS, UBD/PBR and LID domains to control lincRNA-p21 expression. (a) A comparison of ING1 domains and a representation of the ING1b deletion constructs that were used in this study. (b, c and d) Hs68 fibroblasts were treated with either an empty control vector or a deletion mutant of ING1b lacking the plant homeo domain (PHD), nuclear localization signal (NLS), ubiquitin-binding domain/polybasic region (UBD/PBR) or lamin interaction domain (LID) for 48 h before collection of RNA and quantification of lincRNA-p21 expression by qRT-PCR. RNA levels were normalized to GAPDH expression and compared with controls. ING1b, p53 and α-tubulin protein expression levels were measured using western blot analysis. All experiments were carried out in triplicate and asterisks indicate P<0.05)

Mentions: The ING1 gene encodes two major variants with ING1b being the dominant isoform in young (low passage) primary cells, whereas ING1a increases significantly at high-passage levels.10 To test if ING1a could also affect lincRNA-p21 expression, we overexpressed ING1a and subsequently checked lincRNA-p21 levels. Similar to effects seen with overexpression of ING1b, lincRNA-p21 also increased in response to ING1a overexpression (Supplementary Figure S3A). Interestingly, high-passage (senescent) cells also had high basal levels of lincRNA-p21 (Supplementary Figure S3B). Furthermore, lincRNA-p21 was significantly reduced in HGPS cells, a cell type known to have reduced ING1a and ING1b,26 and high levels of p53 expression27, 28, 29 (Supplementary Figure S3C). As shown in Figure 5a, the C-terminal region encompassing the lamin interaction domain (LID), nuclear localization sequence (NLS), PHD and the polybasic region/ubiquitin-binding domain (PBR/UBD) of ING1a and ING1b are identical. The PBR, which activates ING1 and ING2 by binding stress-induced phospholipids,30, 31 overlaps with the UBD, resulting in competition between ubiquitin and phospholipids for binding of this region.14


LincRNA-p21 acts as a mediator of ING1b-induced apoptosis.

Tran UM, Rajarajacholan U, Soh J, Kim TS, Thalappilly S, Sensen CW, Riabowol K - Cell Death Dis (2015)

ING1b requires the NLS, UBD/PBR and LID domains to control lincRNA-p21 expression. (a) A comparison of ING1 domains and a representation of the ING1b deletion constructs that were used in this study. (b, c and d) Hs68 fibroblasts were treated with either an empty control vector or a deletion mutant of ING1b lacking the plant homeo domain (PHD), nuclear localization signal (NLS), ubiquitin-binding domain/polybasic region (UBD/PBR) or lamin interaction domain (LID) for 48 h before collection of RNA and quantification of lincRNA-p21 expression by qRT-PCR. RNA levels were normalized to GAPDH expression and compared with controls. ING1b, p53 and α-tubulin protein expression levels were measured using western blot analysis. All experiments were carried out in triplicate and asterisks indicate P<0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385912&req=5

fig5: ING1b requires the NLS, UBD/PBR and LID domains to control lincRNA-p21 expression. (a) A comparison of ING1 domains and a representation of the ING1b deletion constructs that were used in this study. (b, c and d) Hs68 fibroblasts were treated with either an empty control vector or a deletion mutant of ING1b lacking the plant homeo domain (PHD), nuclear localization signal (NLS), ubiquitin-binding domain/polybasic region (UBD/PBR) or lamin interaction domain (LID) for 48 h before collection of RNA and quantification of lincRNA-p21 expression by qRT-PCR. RNA levels were normalized to GAPDH expression and compared with controls. ING1b, p53 and α-tubulin protein expression levels were measured using western blot analysis. All experiments were carried out in triplicate and asterisks indicate P<0.05)
Mentions: The ING1 gene encodes two major variants with ING1b being the dominant isoform in young (low passage) primary cells, whereas ING1a increases significantly at high-passage levels.10 To test if ING1a could also affect lincRNA-p21 expression, we overexpressed ING1a and subsequently checked lincRNA-p21 levels. Similar to effects seen with overexpression of ING1b, lincRNA-p21 also increased in response to ING1a overexpression (Supplementary Figure S3A). Interestingly, high-passage (senescent) cells also had high basal levels of lincRNA-p21 (Supplementary Figure S3B). Furthermore, lincRNA-p21 was significantly reduced in HGPS cells, a cell type known to have reduced ING1a and ING1b,26 and high levels of p53 expression27, 28, 29 (Supplementary Figure S3C). As shown in Figure 5a, the C-terminal region encompassing the lamin interaction domain (LID), nuclear localization sequence (NLS), PHD and the polybasic region/ubiquitin-binding domain (PBR/UBD) of ING1a and ING1b are identical. The PBR, which activates ING1 and ING2 by binding stress-induced phospholipids,30, 31 overlaps with the UBD, resulting in competition between ubiquitin and phospholipids for binding of this region.14

Bottom Line: We found that this function of lincRNA-p21 is conserved in human cell models.However, their effects become more additive under conditions of stress.Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Oncology, University of Calgary, 3330 Hospital Drive NW, Calgary, T2N 4N1 Alberta, Canada.

ABSTRACT
ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions.

Show MeSH
Related in: MedlinePlus