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Acetylation of C/EBP α inhibits its granulopoietic function

View Article: PubMed Central - PubMed

ABSTRACT

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation.

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Acetylation in the basic region impairs DNA binding ability.(a) GCN5 attenuates DNA binding ability of C/EBPα in vitro. EMSA was performed using a double-stranded C/EBPα binding site oligonucleotide. In vitro-translated C/EBPα was incubated with two separate sources of recombinant GCN5 (HAT) domain proteins. Co-incubation of ivt C/EBPα with GCN5 (lane 6, 7) resulted in decrease in DNA binding as compared to C/EBPα alone (lane 4). No binding was observed from ivt. Empty vector (EV) control (lane 2) or from EV incubated with GCN5 (lane 3). Specificity of C/EBPα binding to the probe was shown by supershift (SS) using C/EBPα antibody (lane 5). (b) Western blot showing in vitro-translated C/EBPα acetylation at K298 and K302 by recombinant GCN5 (HAT domain) protein used in EMSA in a. (c) C/EBPα acetylation mimetic (K2Q-ER) showed reduced DNA binding affinity in EMSA. Equal amounts of nuclear extracts from K562 lines stably transfected with EV-ER (lanes 2, 3), C/EBPα WT-ER (lanes 4, 5), C/EBPα K2Q-ER (lane 6, 7), and C/EBPα K2R-ER (lane 8, 9) were used. Cells were treated with 5 μM β-estradiol for 45 min. Lane 1 contained probe only. In lanes 3, 5, 7 and 9, 1 μL of a supershifting C/EBPα antibody was added. SS indicates supershifted complex; Shift indicates C/EBPα complex; and X refers to nonspecific complex observed with this probe. The representative experiment out of three is shown here. (d) Acetylation mimetic form K2Q was not enriched at a C/EBPα target gene locus G-CSFR27. ChIP analysis of stimulated (45 min) K562 EV-ER, C/EBPα WT-ER, K2Q-ER, and K2R-ER cells using ER antibody. Fold enrichment is calculated compared to binding to control gene, the inactive involucrin (IVL) locus. Data are mean±s.d. (N=3). *P<0.05; Student's unpaired t-test. P value between C/EBPα WT and K2R, K326R, and K326Q is not significant. (e) Model hypothesizing the effect of C/EBPα acetylation on its function in normal hematopoiesis and leukaemia. Non-acetylated C/EBPα is capable of inducing granulocytic differentiation. C/EBPα acetylation leads to loss in DNA binding and loss of recruitment to C/EBPα target genes such as the G-CSF receptor, thereby inhibiting differentiation.
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f5: Acetylation in the basic region impairs DNA binding ability.(a) GCN5 attenuates DNA binding ability of C/EBPα in vitro. EMSA was performed using a double-stranded C/EBPα binding site oligonucleotide. In vitro-translated C/EBPα was incubated with two separate sources of recombinant GCN5 (HAT) domain proteins. Co-incubation of ivt C/EBPα with GCN5 (lane 6, 7) resulted in decrease in DNA binding as compared to C/EBPα alone (lane 4). No binding was observed from ivt. Empty vector (EV) control (lane 2) or from EV incubated with GCN5 (lane 3). Specificity of C/EBPα binding to the probe was shown by supershift (SS) using C/EBPα antibody (lane 5). (b) Western blot showing in vitro-translated C/EBPα acetylation at K298 and K302 by recombinant GCN5 (HAT domain) protein used in EMSA in a. (c) C/EBPα acetylation mimetic (K2Q-ER) showed reduced DNA binding affinity in EMSA. Equal amounts of nuclear extracts from K562 lines stably transfected with EV-ER (lanes 2, 3), C/EBPα WT-ER (lanes 4, 5), C/EBPα K2Q-ER (lane 6, 7), and C/EBPα K2R-ER (lane 8, 9) were used. Cells were treated with 5 μM β-estradiol for 45 min. Lane 1 contained probe only. In lanes 3, 5, 7 and 9, 1 μL of a supershifting C/EBPα antibody was added. SS indicates supershifted complex; Shift indicates C/EBPα complex; and X refers to nonspecific complex observed with this probe. The representative experiment out of three is shown here. (d) Acetylation mimetic form K2Q was not enriched at a C/EBPα target gene locus G-CSFR27. ChIP analysis of stimulated (45 min) K562 EV-ER, C/EBPα WT-ER, K2Q-ER, and K2R-ER cells using ER antibody. Fold enrichment is calculated compared to binding to control gene, the inactive involucrin (IVL) locus. Data are mean±s.d. (N=3). *P<0.05; Student's unpaired t-test. P value between C/EBPα WT and K2R, K326R, and K326Q is not significant. (e) Model hypothesizing the effect of C/EBPα acetylation on its function in normal hematopoiesis and leukaemia. Non-acetylated C/EBPα is capable of inducing granulocytic differentiation. C/EBPα acetylation leads to loss in DNA binding and loss of recruitment to C/EBPα target genes such as the G-CSF receptor, thereby inhibiting differentiation.

Mentions: To investigate whether acetylation by GCN5 alters C/EBPα DNA binding in vitro, we performed electrophoretic mobility shift assays (EMSA). C/EBPα was acetylated by an in vitro acetyltransferase assay using GCN5 HAT domain protein. EMSA analysis showed that acetylation of in vitro-translated C/EBPα reduces its ability to bind to the C/EBPα binding site (Fig. 5a). No binding was observed in the control lysate. Western blot analysis of the reaction used for EMSA confirmed C/EBPα acetylation at K298 and K302 residues (Fig. 5b). MD simulations predicted a dramatic reduction in the ability of C/EBPα to bind to its target DNA sequences by the acetylation mimetic C/EBPα-K2Q (Fig. 3c, Table 1). The DNA-binding abilities of WT and mutated forms of C/EBPα were also assessed by EMSA, using nuclear extracts from K562 lines expressing ER empty vector, C/EBPα WT-ER, C/EBPα K2Q-ER and C/EBPα K2R-ER. As expected, C/EBPα K2Q-ER extracts demonstrated weakened DNA binding as compared with C/EBPα WT-ER and C/EBPα K2R-ER. Using anti-C/EBPα antibody for supershift, we demonstrated specificity of the C/EBPα-dependent binding in EMSA (Fig. 5c and Supplementary Fig. 5f). EMSA performed using 293T cells transfected with C/EBPα K2Q without the ER-tag showed loss of DNA binding similar to K2Q-ER (Supplementary Fig. 5e,g). The G-CSF receptor is an important downstream target gene of C/EBPα, and its promoter region contains C/EBPα-binding sites28. Minimal recruitment of C/EBPα K2Q-ER to the G-CSF receptor promoter by chromatin immunoprecipitation (ChIP) was observed (Fig. 5d). In summary, these results support the hypothesis that acetylation of these two residues leads to the loss of DNA-binding activity and subsequent loss of induction of granulocytic differentiation.


Acetylation of C/EBP α inhibits its granulopoietic function
Acetylation in the basic region impairs DNA binding ability.(a) GCN5 attenuates DNA binding ability of C/EBPα in vitro. EMSA was performed using a double-stranded C/EBPα binding site oligonucleotide. In vitro-translated C/EBPα was incubated with two separate sources of recombinant GCN5 (HAT) domain proteins. Co-incubation of ivt C/EBPα with GCN5 (lane 6, 7) resulted in decrease in DNA binding as compared to C/EBPα alone (lane 4). No binding was observed from ivt. Empty vector (EV) control (lane 2) or from EV incubated with GCN5 (lane 3). Specificity of C/EBPα binding to the probe was shown by supershift (SS) using C/EBPα antibody (lane 5). (b) Western blot showing in vitro-translated C/EBPα acetylation at K298 and K302 by recombinant GCN5 (HAT domain) protein used in EMSA in a. (c) C/EBPα acetylation mimetic (K2Q-ER) showed reduced DNA binding affinity in EMSA. Equal amounts of nuclear extracts from K562 lines stably transfected with EV-ER (lanes 2, 3), C/EBPα WT-ER (lanes 4, 5), C/EBPα K2Q-ER (lane 6, 7), and C/EBPα K2R-ER (lane 8, 9) were used. Cells were treated with 5 μM β-estradiol for 45 min. Lane 1 contained probe only. In lanes 3, 5, 7 and 9, 1 μL of a supershifting C/EBPα antibody was added. SS indicates supershifted complex; Shift indicates C/EBPα complex; and X refers to nonspecific complex observed with this probe. The representative experiment out of three is shown here. (d) Acetylation mimetic form K2Q was not enriched at a C/EBPα target gene locus G-CSFR27. ChIP analysis of stimulated (45 min) K562 EV-ER, C/EBPα WT-ER, K2Q-ER, and K2R-ER cells using ER antibody. Fold enrichment is calculated compared to binding to control gene, the inactive involucrin (IVL) locus. Data are mean±s.d. (N=3). *P<0.05; Student's unpaired t-test. P value between C/EBPα WT and K2R, K326R, and K326Q is not significant. (e) Model hypothesizing the effect of C/EBPα acetylation on its function in normal hematopoiesis and leukaemia. Non-acetylated C/EBPα is capable of inducing granulocytic differentiation. C/EBPα acetylation leads to loss in DNA binding and loss of recruitment to C/EBPα target genes such as the G-CSF receptor, thereby inhibiting differentiation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4814574&req=5

f5: Acetylation in the basic region impairs DNA binding ability.(a) GCN5 attenuates DNA binding ability of C/EBPα in vitro. EMSA was performed using a double-stranded C/EBPα binding site oligonucleotide. In vitro-translated C/EBPα was incubated with two separate sources of recombinant GCN5 (HAT) domain proteins. Co-incubation of ivt C/EBPα with GCN5 (lane 6, 7) resulted in decrease in DNA binding as compared to C/EBPα alone (lane 4). No binding was observed from ivt. Empty vector (EV) control (lane 2) or from EV incubated with GCN5 (lane 3). Specificity of C/EBPα binding to the probe was shown by supershift (SS) using C/EBPα antibody (lane 5). (b) Western blot showing in vitro-translated C/EBPα acetylation at K298 and K302 by recombinant GCN5 (HAT domain) protein used in EMSA in a. (c) C/EBPα acetylation mimetic (K2Q-ER) showed reduced DNA binding affinity in EMSA. Equal amounts of nuclear extracts from K562 lines stably transfected with EV-ER (lanes 2, 3), C/EBPα WT-ER (lanes 4, 5), C/EBPα K2Q-ER (lane 6, 7), and C/EBPα K2R-ER (lane 8, 9) were used. Cells were treated with 5 μM β-estradiol for 45 min. Lane 1 contained probe only. In lanes 3, 5, 7 and 9, 1 μL of a supershifting C/EBPα antibody was added. SS indicates supershifted complex; Shift indicates C/EBPα complex; and X refers to nonspecific complex observed with this probe. The representative experiment out of three is shown here. (d) Acetylation mimetic form K2Q was not enriched at a C/EBPα target gene locus G-CSFR27. ChIP analysis of stimulated (45 min) K562 EV-ER, C/EBPα WT-ER, K2Q-ER, and K2R-ER cells using ER antibody. Fold enrichment is calculated compared to binding to control gene, the inactive involucrin (IVL) locus. Data are mean±s.d. (N=3). *P<0.05; Student's unpaired t-test. P value between C/EBPα WT and K2R, K326R, and K326Q is not significant. (e) Model hypothesizing the effect of C/EBPα acetylation on its function in normal hematopoiesis and leukaemia. Non-acetylated C/EBPα is capable of inducing granulocytic differentiation. C/EBPα acetylation leads to loss in DNA binding and loss of recruitment to C/EBPα target genes such as the G-CSF receptor, thereby inhibiting differentiation.
Mentions: To investigate whether acetylation by GCN5 alters C/EBPα DNA binding in vitro, we performed electrophoretic mobility shift assays (EMSA). C/EBPα was acetylated by an in vitro acetyltransferase assay using GCN5 HAT domain protein. EMSA analysis showed that acetylation of in vitro-translated C/EBPα reduces its ability to bind to the C/EBPα binding site (Fig. 5a). No binding was observed in the control lysate. Western blot analysis of the reaction used for EMSA confirmed C/EBPα acetylation at K298 and K302 residues (Fig. 5b). MD simulations predicted a dramatic reduction in the ability of C/EBPα to bind to its target DNA sequences by the acetylation mimetic C/EBPα-K2Q (Fig. 3c, Table 1). The DNA-binding abilities of WT and mutated forms of C/EBPα were also assessed by EMSA, using nuclear extracts from K562 lines expressing ER empty vector, C/EBPα WT-ER, C/EBPα K2Q-ER and C/EBPα K2R-ER. As expected, C/EBPα K2Q-ER extracts demonstrated weakened DNA binding as compared with C/EBPα WT-ER and C/EBPα K2R-ER. Using anti-C/EBPα antibody for supershift, we demonstrated specificity of the C/EBPα-dependent binding in EMSA (Fig. 5c and Supplementary Fig. 5f). EMSA performed using 293T cells transfected with C/EBPα K2Q without the ER-tag showed loss of DNA binding similar to K2Q-ER (Supplementary Fig. 5e,g). The G-CSF receptor is an important downstream target gene of C/EBPα, and its promoter region contains C/EBPα-binding sites28. Minimal recruitment of C/EBPα K2Q-ER to the G-CSF receptor promoter by chromatin immunoprecipitation (ChIP) was observed (Fig. 5d). In summary, these results support the hypothesis that acetylation of these two residues leads to the loss of DNA-binding activity and subsequent loss of induction of granulocytic differentiation.

View Article: PubMed Central - PubMed

ABSTRACT

CCAAT/enhancer-binding protein alpha (C/EBP&alpha;) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBP&alpha; at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBP&alpha; DNA-binding ability and modulates C/EBP&alpha; transcriptional activity. Acetylated C/EBP&alpha; is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBP&alpha; mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBP&alpha;-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBP&alpha; and demonstrate the importance of C/EBP&alpha; acetylation in myeloid differentiation.

No MeSH data available.


Related in: MedlinePlus