Limits...
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.

Show MeSH

Related in: MedlinePlus

NF-YB silencing induces apoptosis via caspases activation. (A) Cell cycle distribution analysis via flow cytometry of non-targeting control and NF-YB siRNA transfected cells, untreated or treated with ZVAD. (B) Western blot analysis of negative control and NF-YB-silenced HCT116 total extracts with anti-NF-YB and anti-actin antibodies, untreated or treated with ZVAD. (C) Expression analysis (RT-PCR) of the indicated caspase mRNA transcripts. (D) Immunofluorescence analysis of the indicated cleaved caspases and HOECHST.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275158&req=5

Figure 5: NF-YB silencing induces apoptosis via caspases activation. (A) Cell cycle distribution analysis via flow cytometry of non-targeting control and NF-YB siRNA transfected cells, untreated or treated with ZVAD. (B) Western blot analysis of negative control and NF-YB-silenced HCT116 total extracts with anti-NF-YB and anti-actin antibodies, untreated or treated with ZVAD. (C) Expression analysis (RT-PCR) of the indicated caspase mRNA transcripts. (D) Immunofluorescence analysis of the indicated cleaved caspases and HOECHST.

Mentions: To explain the role of NF-Y inactivation in the induction of apoptosis, we turned our attention to caspases, because their cleavage and activation play a central role in the initiation and execution of this process. We first examined whether apoptosis is a caspase-dependent phenomenon in NF-YB-inactivated HCT116, treating them with the broad-spectrum caspase inhibitor Z-VAD. Figure 5A shows that apoptosis was significantly reduced in the presence of Z-VAD. A dramatic decrease in sub-G1 cells is indeed observed, associated to a modest increase in G1 cells. Western blot analysis confirmed that NF-YB protein levels were drastically reduced both in untreated and in Z-VAD-treated cells (Figure 5B), ruling out that the lack of apoptosis is due to inefficient NF-YB inactivation.Figure 5.


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 silencing induces apoptosis via caspases activation. (A) Cell cycle distribution analysis via flow cytometry of non-targeting control and NF-YB siRNA transfected cells, untreated or treated with ZVAD. (B) Western blot analysis of negative control and NF-YB-silenced HCT116 total extracts with anti-NF-YB and anti-actin antibodies, untreated or treated with ZVAD. (C) Expression analysis (RT-PCR) of the indicated caspase mRNA transcripts. (D) Immunofluorescence analysis of the indicated cleaved caspases and HOECHST.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275158&req=5

Figure 5: NF-YB silencing induces apoptosis via caspases activation. (A) Cell cycle distribution analysis via flow cytometry of non-targeting control and NF-YB siRNA transfected cells, untreated or treated with ZVAD. (B) Western blot analysis of negative control and NF-YB-silenced HCT116 total extracts with anti-NF-YB and anti-actin antibodies, untreated or treated with ZVAD. (C) Expression analysis (RT-PCR) of the indicated caspase mRNA transcripts. (D) Immunofluorescence analysis of the indicated cleaved caspases and HOECHST.
Mentions: To explain the role of NF-Y inactivation in the induction of apoptosis, we turned our attention to caspases, because their cleavage and activation play a central role in the initiation and execution of this process. We first examined whether apoptosis is a caspase-dependent phenomenon in NF-YB-inactivated HCT116, treating them with the broad-spectrum caspase inhibitor Z-VAD. Figure 5A shows that apoptosis was significantly reduced in the presence of Z-VAD. A dramatic decrease in sub-G1 cells is indeed observed, associated to a modest increase in G1 cells. Western blot analysis confirmed that NF-YB protein levels were drastically reduced both in untreated and in Z-VAD-treated cells (Figure 5B), ruling out that the lack of apoptosis is due to inefficient NF-YB inactivation.Figure 5.

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