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A balance between NF-Y and p53 governs the pro- and anti-apoptotic transcriptional response.

Benatti P, Basile V, Merico D, Fantoni LI, Tagliafico E, Imbriano C - Nucleic Acids Res. (2008)

Bottom Line: In this study, we examined the functional and molecular effects of NF-YB knockdown.Failure to maintain a physiologic level of CCAAT-dependent transcription of anti-apoptotic genes contributes to impairment of Bax/Bcl-2 and Bax/Bcl-X(L) ratios.Our data highlight the importance of fine balancing the NF-Y-p53 duo for cell survival by (i) maintaining transcription of anti-apoptotic genes and (ii) preventing p53 activation that triggers the apoptotic cascade.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia Animale, Università di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy.

ABSTRACT
The transcription factor NF-Y is a trimer with histone-like subunits that binds and activates CCAAT-containing promoters. NF-Y controls the expression of several key regulators of the cell cycle. In this study, we examined the functional and molecular effects of NF-YB knockdown. Cell cycle progression is affected with a G2/M-specific depletion. This is due to the inability of activation of G2/M-specific genes, as evidenced by expression profiling, RT-PCR and ChIP data. Surprisingly, apoptosis is also observed, with Caspase 3/7/8 cleavage. A role of p53 and Bcl-2 family members is important. NF-YB inactivation is sufficient to functionally activate p53, in the absence of DNA damage. Failure to maintain a physiologic level of CCAAT-dependent transcription of anti-apoptotic genes contributes to impairment of Bax/Bcl-2 and Bax/Bcl-X(L) ratios. Our data highlight the importance of fine balancing the NF-Y-p53 duo for cell survival by (i) maintaining transcription of anti-apoptotic genes and (ii) preventing p53 activation that triggers the apoptotic cascade.

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Related in: MedlinePlus

NF-YB RNAi in HCT116 cells. (A) Three siRNA were designed on human NF-YB gene. Black boxes represent exons; red boxes indicate targeted exons. (B) Left panel: RT-PCR analysis of NF-YB and GAPDH mRNA transcripts in negative control and NF-YB1-2-3-silenced cells. Right panel: western blot analysis of HCT116 total extracts with anti-NF-YB and anti-actin antibodies, transfected with control and NF-YB siRNAs. (C) RT-PCR (left panel) and western blot analysis (right panel) of the three NF-Y subunits in control or NF-YB2 transfected cells. (D) Chromatin immunoprecipitation analysis of NF-Y targets, using control and NF-YB-silenced HCT116 cells with NF-YB and Flag antibodies.
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Figure 1: NF-YB RNAi in HCT116 cells. (A) Three siRNA were designed on human NF-YB gene. Black boxes represent exons; red boxes indicate targeted exons. (B) Left panel: RT-PCR analysis of NF-YB and GAPDH mRNA transcripts in negative control and NF-YB1-2-3-silenced cells. Right panel: western blot analysis of HCT116 total extracts with anti-NF-YB and anti-actin antibodies, transfected with control and NF-YB siRNAs. (C) RT-PCR (left panel) and western blot analysis (right panel) of the three NF-Y subunits in control or NF-YB2 transfected cells. (D) Chromatin immunoprecipitation analysis of NF-Y targets, using control and NF-YB-silenced HCT116 cells with NF-YB and Flag antibodies.

Mentions: We designed three RNA-interference (RNAi) oligos to disrupt the function of the NF-YB subunit (Figure 1A); transfections of HCT116 cells with two of these siRNAs —NF-YB2 and NF-YB3— resulted in a significant and reproducible degradation of NF-YB mRNA, as shown by RT-PCR analysis (Figure 1B, left panel), and protein, as judged by western blotting (Figure 1B, right panel). The degree of knockdown was 75–95%. The actin protein and GAPDH mRNA were used as internal controls (Figure 1B, right and left panels, respectively). We analyzed the effect of NF-YB silencing on the transcription and expression of the other NF-Y subunits. We noticed increased transcription of NF-YC, and no changes in the two NF-YA isoforms, as shown by RT-PCR analysis (Figure 1C, left panel). Consistent with this, western blots showed increased NF-YC levels, and no change for NF-YA (Figure 1C, right panel and Supplementary Figure 1A). Finally, to ascertain whether NF-Y binding was affected in RNAi inactivated cells, ChIP assays with the anti-NF-YB antibody were performed. As shown in Figure 1D, NF-YB siRNA reduces considerably the binding to CCNB2, Cdc2, Cdc25C and TK promoters. The PCR of each ChIP were quantitatively compared to a Flag control antibody (Supplementary Figure 2A). We conclude that effective inactivation of NF-YB can be achieved in HCT116 cells.Figure 1.


A balance between NF-Y and p53 governs the pro- and anti-apoptotic transcriptional response.

Benatti P, Basile V, Merico D, Fantoni LI, Tagliafico E, Imbriano C - Nucleic Acids Res. (2008)

NF-YB RNAi in HCT116 cells. (A) Three siRNA were designed on human NF-YB gene. Black boxes represent exons; red boxes indicate targeted exons. (B) Left panel: RT-PCR analysis of NF-YB and GAPDH mRNA transcripts in negative control and NF-YB1-2-3-silenced cells. Right panel: western blot analysis of HCT116 total extracts with anti-NF-YB and anti-actin antibodies, transfected with control and NF-YB siRNAs. (C) RT-PCR (left panel) and western blot analysis (right panel) of the three NF-Y subunits in control or NF-YB2 transfected cells. (D) Chromatin immunoprecipitation analysis of NF-Y targets, using control and NF-YB-silenced HCT116 cells with NF-YB and Flag antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: NF-YB RNAi in HCT116 cells. (A) Three siRNA were designed on human NF-YB gene. Black boxes represent exons; red boxes indicate targeted exons. (B) Left panel: RT-PCR analysis of NF-YB and GAPDH mRNA transcripts in negative control and NF-YB1-2-3-silenced cells. Right panel: western blot analysis of HCT116 total extracts with anti-NF-YB and anti-actin antibodies, transfected with control and NF-YB siRNAs. (C) RT-PCR (left panel) and western blot analysis (right panel) of the three NF-Y subunits in control or NF-YB2 transfected cells. (D) Chromatin immunoprecipitation analysis of NF-Y targets, using control and NF-YB-silenced HCT116 cells with NF-YB and Flag antibodies.
Mentions: We designed three RNA-interference (RNAi) oligos to disrupt the function of the NF-YB subunit (Figure 1A); transfections of HCT116 cells with two of these siRNAs —NF-YB2 and NF-YB3— resulted in a significant and reproducible degradation of NF-YB mRNA, as shown by RT-PCR analysis (Figure 1B, left panel), and protein, as judged by western blotting (Figure 1B, right panel). The degree of knockdown was 75–95%. The actin protein and GAPDH mRNA were used as internal controls (Figure 1B, right and left panels, respectively). We analyzed the effect of NF-YB silencing on the transcription and expression of the other NF-Y subunits. We noticed increased transcription of NF-YC, and no changes in the two NF-YA isoforms, as shown by RT-PCR analysis (Figure 1C, left panel). Consistent with this, western blots showed increased NF-YC levels, and no change for NF-YA (Figure 1C, right panel and Supplementary Figure 1A). Finally, to ascertain whether NF-Y binding was affected in RNAi inactivated cells, ChIP assays with the anti-NF-YB antibody were performed. As shown in Figure 1D, NF-YB siRNA reduces considerably the binding to CCNB2, Cdc2, Cdc25C and TK promoters. The PCR of each ChIP were quantitatively compared to a Flag control antibody (Supplementary Figure 2A). We conclude that effective inactivation of NF-YB can be achieved in HCT116 cells.Figure 1.

Bottom Line: In this study, we examined the functional and molecular effects of NF-YB knockdown.Failure to maintain a physiologic level of CCAAT-dependent transcription of anti-apoptotic genes contributes to impairment of Bax/Bcl-2 and Bax/Bcl-X(L) ratios.Our data highlight the importance of fine balancing the NF-Y-p53 duo for cell survival by (i) maintaining transcription of anti-apoptotic genes and (ii) preventing p53 activation that triggers the apoptotic cascade.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia Animale, Università di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy.

ABSTRACT
The transcription factor NF-Y is a trimer with histone-like subunits that binds and activates CCAAT-containing promoters. NF-Y controls the expression of several key regulators of the cell cycle. In this study, we examined the functional and molecular effects of NF-YB knockdown. Cell cycle progression is affected with a G2/M-specific depletion. This is due to the inability of activation of G2/M-specific genes, as evidenced by expression profiling, RT-PCR and ChIP data. Surprisingly, apoptosis is also observed, with Caspase 3/7/8 cleavage. A role of p53 and Bcl-2 family members is important. NF-YB inactivation is sufficient to functionally activate p53, in the absence of DNA damage. Failure to maintain a physiologic level of CCAAT-dependent transcription of anti-apoptotic genes contributes to impairment of Bax/Bcl-2 and Bax/Bcl-X(L) ratios. Our data highlight the importance of fine balancing the NF-Y-p53 duo for cell survival by (i) maintaining transcription of anti-apoptotic genes and (ii) preventing p53 activation that triggers the apoptotic cascade.

Show MeSH
Related in: MedlinePlus