Posttranslational regulation of NF-YA modulates NF-Y transcriptional activity

Abstract

NF-Y binds to CCAAT motifs in the promoter region of a variety of genes involved in cell cycle progression. The NF-Y complex comprises three subunits, NF-YA, -YB, and -YC, all required for DNA binding. Expression of NF-YA fluctuates during the cell cycle and is down-regulated in postmitotic cells, indicating its role as the regulatory subunit of the complex. Control of NF-YA accumulation is posttranscriptional, NF-YA mRNA being relatively constant. Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity.

Figure 1.

The proteasome pathway regulates NF-YA expression. (A) Western blot analysis of endogenous NF-YA, -YB, and -YC in nuclear and cytoplasmic extracts from myoblasts and myotubes treated with 50 μM LLnL for 2, 4, and 8 h. (B) Western blot analysis of the endogenous NF-YA, -YB, -YC, and p53 in nuclear and cytoplasmic extracts from myoblasts treated with 50 μM LLM or MG132 for 4 and 8 h.

Figure 2.

The half-life of NF-YA is increase by proteasome inhibition. (A) Densitometric analysis of the NF-YA or -YB as a control expression level after normalization against the values obtained for the control housekeeping gene tubulin under the same experimental conditions. (B) Western blot analysis of the endogenous NF-YA and -YB, in total extracts from C2C12 cells treated with 100 μg/ml Chx for various lengths of time and coincubated with 100 μg/ml Chx and 50 μM of LLnL. α-tubulin is used as loading control.

Figure 3.

Exogenous NF-YA protein is ubiquitylated in vivo. C2C12 cells were transfected with plasmids encoding NF-YA (A–C) or -YB (D) together with UbHA. Total extracts from these cells were immunoprecipitated with increasing amounts of anti-NF-YA (A and B), anti-HA (C) or anti-NF-YB antibodies (D). Western blot analysis was performed with an anti-HA antibody in (A and D) or anti NF-YA antibody (C and B). In all immunoprecipitation experiments an appropriate control antibody was used. Stars indicate the antibody chains used in the immunoprecipitations (IPs).

Figure 4.

Endogenous NF-YA protein is ubiquitylated in vivo. (A) Immunoprecipitation of endogenous NF-YA from total extracts from C2C12 cells with anti-NF-YA antibody and Western blot analysis with the ubiquitin-specific antibody FK2 (α Ub). (B) Immunoprecipitation of endogenous NF-YA from total extracts from C2C12 cells with mouse monoclonal anti-NF-YA antibody and Western blot analysis with the anti-NF-YA rabbit polyclonal antibody. (C) Immunoprecipitation of endogenous ubiquitylated proteins from total extracts from C2C12 cells with anti-FK2 antibody and Western blot analysis with anti-NF-YA antibody. In all immunoprecipitation experiments an appropriate control antibody was used. Stars indicate the antibody chains used in the immunoprecipitations (IPs).

Figure 5.

Identification of NF-YA lysine residues targets for ubiquitylation. (A) Schematic representation of the C-terminus domain of the NF-YA protein containing six lysine residues conserved across species. (B) Schematic representation of NF-YA protein with the positions of the lysines replaced in the different NF-YA mutants used in this work. (C) Number of GFP-positive cells in cell populations transfected with mutants (YA-R1, -R2, and YA-R3), wild-type NF-YA (NF-YA), or the empty vector as a control, at different times. (D) Time course of wild-type and mutants NF-YA expression by Western blot analysis on total extracts from cells nontransfected (NT) or transfected with mutants (YA-R1, -R2, and -R3) or wild-type NF-YA (NF-YA).

Figure 6.

Identification of NF-YA lysine residues targets for ubiquitylation. (A) Western blot analysis on total cell extracts from nontransfected C2C12 cells (NT) and cells transfected with increasing amounts of plasmids encoding wild-type (NF-YA) or mutant NF-YA (NF-YA R2÷R3; see Figure 5B). (B) Total extracts from cells overexpressing wild-type or YA-R1, -R2, -R3, and -R2÷R3, immunoprecipitated with anti-NF-YA antibody, or mouse serum. Western blot analysis was performed with an anti-ubiquitin antibody FK2 (α Ub). In all immunoprecipitation experiments an appropriate control antibody was used. Stars indicate the antibody chains used in the immunoprecipitations (IPs). (C) Densitometric analysis of NF-YA ubiquitin ladder.

Figure 7.

p300 acetylation of NF-YA in vitro. (A) In vitro acetylation by p300 of the NF-YA proteins outlined in the scheme. YA9 harbors only the highly conserved part of NF-YA. (B) EMSA analysis of wild-type NF-YA (NF-YA) and YA-R1, -R2, and -R3 mutants on a CCAAT-box containing oligonucleotide. A dose-response 0.1, 0.3, and 1 ng of purified NF-YAs was preincubated with recombinant NF-YB and -YC, 5 ng, and added to labeled DNA. (C) In vitro acetylation of recombinant purified NF-YA mutants by p300. In the bottom panel, Coomassie blue staining of the SDS gel is shown.

Figure 8.

p300 expression impacts on NF-YA ubiquitylation. (A) Western blot analysis of NF-YA in total cell extracts from C2C12 cells transfected with p300. Actin was used as loading control. (B) C2C12 cells were transfected with wild-type NF-YA and increasing amounts of p300. Total extracts from these cells were immunoprecipitated with anti-NF-YA antibody and Western blot analysis with the ubiquitin-specific antibody, FK2 (α Ub). In immunoprecipitation experiments an appropriate control antibody was used. Stars indicate the antibody chains used in the IPs. (C) Western blot analysis of the endogenous NF-YA and p300 in total extracts from HCT116 cells treated with 100 μg/ml Chx for 2h and transfected with an oligo pool directed against p300 (sip300) or an unrelated sequence (scramble). Actin was used as loading control. (D) Semiquantitative RT-PCR analysis of NF-YA and p300 expression on total RNA from C2C12 cells transfected with increasing amounts of p300. Aldolase was used as loading control. Ethidium bromide staining of total RNA is shown. Stars indicate the antibody chains used in the immunoprecipitations (IPs).

Figure 9.

Acetylation and ubiquitylation of NF-YA protein in pre- and postmitotic cells. (A) Immunoprecipitation of endogenous NF-YA from nuclear (NE) and cytoplasmic (CE) extracts of myoblasts with anti-NF-YA antibody and Western blot analysis with ubiquitin-specific antibody FK2 (α Ub) and anti-acetyl-lysine (α Acetil-lys). (B) Immunoprecipitation of endogenous NF-YA from nuclear extracts of myoblasts after treatment with 50 μM of LLnL with anti-NF-YA antibody and Western blot analysis with α Ub and α Acetil-lys. (C) Immunoprecipitation of endogenous NF-YA from nuclear extracts of myotubes after treatment with 50 μM of LLnL with anti-NF-YA antibody and Western blot analysis with α Ub and α Acetil-lys. In all panels the amount of immunoprecipitated NF-YA has been measured by Western blot with an anti-NF-YA antibody. In all immunoprecipitation experiments an appropriate control antibody has been used. Stars indicate the antibody chains used in the immunoprecipitations (IPs).

Figure 10.

Acetylation and ubiquitylation of NF-YA lysine residues are involved on its transcriptional activity. (A) Fluorescence of NF-YA GFP and mutant NF-YA (YA-R1, -R2, and -R3) GFP proteins in C2C12 cells. (B) Luciferase activity of C2C12 cells cotransfected with pCCAAT-B2LUC and wild-type NF-YA or its mutants. (C) Luciferase activity of C2C12 cells cotransfected with pCCAAT-B2LUC and wild-type NF-YA and p300 or ubiquitin (UbHA). In both B and C luciferase activity was normalized for β-gal and for protein amount. The data represent the mean ± SD of triplicate determinations from three separate experiments.

Figure 11.

Forced expression of a stable NF-YA protein sustains expression of its target genes and influences cell proliferation. Western blot analysis of NF-YA, cyclin B1, cyclin A, cdk1, and HSP70 as loading control in C2C12 cells at different times posttransfection. Left, C2C12 cells transfected with empty vector. Middle and right, C2C12 cells transfected with plasmids encoding wild-type NF-YA or mutant NF-YA YA-R3 respectively.