Limits...
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.

Show MeSH

Related in: MedlinePlus

ING1b regulates lincRNA-p21 expression, and both ING1b and p53 are required for lincRNA-p21 induction after DNA damage. (a) Following infection of normal human diploid fibroblasts (Hs68) with adenoviral constructs containing GFP (Ad-GFP) or GFP-ING1b (Ad-ING1b), expression of lincRNA-p21 was measured using qRT-PCR, normalized to β-actin and graphed relative to Ad-GFP controls. ING1b, p53 and α-tubulin protein levels were examined using western blot analysis. (b and c) Hs68 cells were transfected with control or ING1b siRNAs for 32 h, and a subset was treated with Adriamycin (ADR) for another 16 h. RNA levels of lincRNA-p21 were measured using qRT-PCR, normalized to GAPDH and graphed relative to controls. ING1b and p53 protein levels were determined using western blot and normalized to α-tubulin protein expression. P53- and p53-induced H1299 cells were transfected with ING1b or control siRNAs for 24 h before treatment with 500 nM ADR for 16 h (d and e), 100 μM hydrogen peroxide (H2O2) for 24 h (f) and 85 J/m2 of UV (24 h) (g). RNA was extracted and lincRNA-p21 was quantified using qRT-PCR. All values in this figure represent an average of three independent experiments (asterisks indicate P<0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: ING1b regulates lincRNA-p21 expression, and both ING1b and p53 are required for lincRNA-p21 induction after DNA damage. (a) Following infection of normal human diploid fibroblasts (Hs68) with adenoviral constructs containing GFP (Ad-GFP) or GFP-ING1b (Ad-ING1b), expression of lincRNA-p21 was measured using qRT-PCR, normalized to β-actin and graphed relative to Ad-GFP controls. ING1b, p53 and α-tubulin protein levels were examined using western blot analysis. (b and c) Hs68 cells were transfected with control or ING1b siRNAs for 32 h, and a subset was treated with Adriamycin (ADR) for another 16 h. RNA levels of lincRNA-p21 were measured using qRT-PCR, normalized to GAPDH and graphed relative to controls. ING1b and p53 protein levels were determined using western blot and normalized to α-tubulin protein expression. P53- and p53-induced H1299 cells were transfected with ING1b or control siRNAs for 24 h before treatment with 500 nM ADR for 16 h (d and e), 100 μM hydrogen peroxide (H2O2) for 24 h (f) and 85 J/m2 of UV (24 h) (g). RNA was extracted and lincRNA-p21 was quantified using qRT-PCR. All values in this figure represent an average of three independent experiments (asterisks indicate P<0.05)

Mentions: Previous studies have reported that ING1b-induced apoptosis can be either p53-dependent or p53-independent, with variations being contingent on cell type.15 Moreover, p53 was shown to require ING1 for induction of programmed cell death,11, 13, 14 suggesting the potential for a functional interdependency between ING1 and p53. Recently, lincRNA-p21 was shown to be a transcriptional target of p53 that mediates gene repression to promote apoptosis.16 Given this relationship between ING1 and p53, we asked if ING1b could influence lincRNA-p21 expression. ING1b was overexpressed using an adenoviral construct encoding GFP and ING1b expressed from different promoters, or knocked down with siRNAs in Hs68 human diploid fibroblasts. Levels of lincRNA-p21 was subsequently examined using quantitative real-time polymerase chain reactions (qRT-PCR). In this human diploid fibroblast strain, ING1b increased lincRNA-p21 levels by ~40-fold (Figure 1a). In contrast, lincRNA-p21 did not decrease in response to ING1b knockdown (Figure 1b), suggesting that ING1b influences lincRNA-p21 expression only at levels that induce cell stress, similar to those during apoptosis.18, 19 As lincRNA-p21 was also reported to respond to p53 downstream of DNA-damage signals,16 we next tested the effects of ING1b on lincRNA-p21 in the presence of damage. Hs68s treated with control oligo or with siING1b were exposed to adriamycin (ADR) to induce DNA damage. Under control conditions, lincRNA-p21 expression increased significantly after DNA damage (Figure 1c, Supplementary Figure S1A), but induction was only 50% as efficient when ING1b was knocked down (Figure 1c, P<0.05), suggesting ING1b was transducing a major part of the ADR stress signal. This was not due to the effects of ING1b on p53 transcription (Supplementary Figure S1B). Instead, we found that this decrease in efficiency but not absolute suppression of lincRNA-p21 expression may be due to an increase in the half-life of lincRNA-p21 after ING1b knockdown. Another factor that might affect the efficiency of knockdown might be differential stabilization of ING1 protein following DNA damage as reported previously for conditions of oxidative stress.20, 21 Compared with control, cells treated with ING1b shRNA exhibited increased lincRNA-p21 stability after DNA damage (Supplementary Figure S1C). In this experiment actinomycin D (ActD) was used to block ongoing transcription to estimate the t1/2 of lincRNA-p21. As ActD functions by binding gene promoters to block transcription and ING1b also binds the promoter region of lincRNA-p21 (see below), knockdown of ING1b might also interfere with the ability of ActD to block transcription, leading to apparent increased lincRNA-p21 stability.


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 regulates lincRNA-p21 expression, and both ING1b and p53 are required for lincRNA-p21 induction after DNA damage. (a) Following infection of normal human diploid fibroblasts (Hs68) with adenoviral constructs containing GFP (Ad-GFP) or GFP-ING1b (Ad-ING1b), expression of lincRNA-p21 was measured using qRT-PCR, normalized to β-actin and graphed relative to Ad-GFP controls. ING1b, p53 and α-tubulin protein levels were examined using western blot analysis. (b and c) Hs68 cells were transfected with control or ING1b siRNAs for 32 h, and a subset was treated with Adriamycin (ADR) for another 16 h. RNA levels of lincRNA-p21 were measured using qRT-PCR, normalized to GAPDH and graphed relative to controls. ING1b and p53 protein levels were determined using western blot and normalized to α-tubulin protein expression. P53- and p53-induced H1299 cells were transfected with ING1b or control siRNAs for 24 h before treatment with 500 nM ADR for 16 h (d and e), 100 μM hydrogen peroxide (H2O2) for 24 h (f) and 85 J/m2 of UV (24 h) (g). RNA was extracted and lincRNA-p21 was quantified using qRT-PCR. All values in this figure represent an average of three independent experiments (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

fig1: ING1b regulates lincRNA-p21 expression, and both ING1b and p53 are required for lincRNA-p21 induction after DNA damage. (a) Following infection of normal human diploid fibroblasts (Hs68) with adenoviral constructs containing GFP (Ad-GFP) or GFP-ING1b (Ad-ING1b), expression of lincRNA-p21 was measured using qRT-PCR, normalized to β-actin and graphed relative to Ad-GFP controls. ING1b, p53 and α-tubulin protein levels were examined using western blot analysis. (b and c) Hs68 cells were transfected with control or ING1b siRNAs for 32 h, and a subset was treated with Adriamycin (ADR) for another 16 h. RNA levels of lincRNA-p21 were measured using qRT-PCR, normalized to GAPDH and graphed relative to controls. ING1b and p53 protein levels were determined using western blot and normalized to α-tubulin protein expression. P53- and p53-induced H1299 cells were transfected with ING1b or control siRNAs for 24 h before treatment with 500 nM ADR for 16 h (d and e), 100 μM hydrogen peroxide (H2O2) for 24 h (f) and 85 J/m2 of UV (24 h) (g). RNA was extracted and lincRNA-p21 was quantified using qRT-PCR. All values in this figure represent an average of three independent experiments (asterisks indicate P<0.05)
Mentions: Previous studies have reported that ING1b-induced apoptosis can be either p53-dependent or p53-independent, with variations being contingent on cell type.15 Moreover, p53 was shown to require ING1 for induction of programmed cell death,11, 13, 14 suggesting the potential for a functional interdependency between ING1 and p53. Recently, lincRNA-p21 was shown to be a transcriptional target of p53 that mediates gene repression to promote apoptosis.16 Given this relationship between ING1 and p53, we asked if ING1b could influence lincRNA-p21 expression. ING1b was overexpressed using an adenoviral construct encoding GFP and ING1b expressed from different promoters, or knocked down with siRNAs in Hs68 human diploid fibroblasts. Levels of lincRNA-p21 was subsequently examined using quantitative real-time polymerase chain reactions (qRT-PCR). In this human diploid fibroblast strain, ING1b increased lincRNA-p21 levels by ~40-fold (Figure 1a). In contrast, lincRNA-p21 did not decrease in response to ING1b knockdown (Figure 1b), suggesting that ING1b influences lincRNA-p21 expression only at levels that induce cell stress, similar to those during apoptosis.18, 19 As lincRNA-p21 was also reported to respond to p53 downstream of DNA-damage signals,16 we next tested the effects of ING1b on lincRNA-p21 in the presence of damage. Hs68s treated with control oligo or with siING1b were exposed to adriamycin (ADR) to induce DNA damage. Under control conditions, lincRNA-p21 expression increased significantly after DNA damage (Figure 1c, Supplementary Figure S1A), but induction was only 50% as efficient when ING1b was knocked down (Figure 1c, P<0.05), suggesting ING1b was transducing a major part of the ADR stress signal. This was not due to the effects of ING1b on p53 transcription (Supplementary Figure S1B). Instead, we found that this decrease in efficiency but not absolute suppression of lincRNA-p21 expression may be due to an increase in the half-life of lincRNA-p21 after ING1b knockdown. Another factor that might affect the efficiency of knockdown might be differential stabilization of ING1 protein following DNA damage as reported previously for conditions of oxidative stress.20, 21 Compared with control, cells treated with ING1b shRNA exhibited increased lincRNA-p21 stability after DNA damage (Supplementary Figure S1C). In this experiment actinomycin D (ActD) was used to block ongoing transcription to estimate the t1/2 of lincRNA-p21. As ActD functions by binding gene promoters to block transcription and ING1b also binds the promoter region of lincRNA-p21 (see below), knockdown of ING1b might also interfere with the ability of ActD to block transcription, leading to apparent increased lincRNA-p21 stability.

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