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Acetylation of C/EBP α inhibits its granulopoietic function

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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.

No MeSH data available.


Molecular dynamics simulation of acetylation at K298 and K302.(a) Diagram of the C/EBPα protein. The positions of the three transactivation elements (TEs), basic region-leucine zipper (BR-LZ) and putative acetylation sites for GCN5 are indicated. (b) The two conformations of the acetylated lysine (ACK) side chain built into the starting coordinates of the acetylated models and preserved during the MD simulations. (c) MD snapshots during 3 ns simulation showing selected protein–DNA interactions: I) C/EBPα WT-DNA; II) K2Q-DNA; III) K2Ac_a-DNA; and IV) K2Ac_b-DNA. Hydrogen bonds are shown as dashed lines.
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f3: Molecular dynamics simulation of acetylation at K298 and K302.(a) Diagram of the C/EBPα protein. The positions of the three transactivation elements (TEs), basic region-leucine zipper (BR-LZ) and putative acetylation sites for GCN5 are indicated. (b) The two conformations of the acetylated lysine (ACK) side chain built into the starting coordinates of the acetylated models and preserved during the MD simulations. (c) MD snapshots during 3 ns simulation showing selected protein–DNA interactions: I) C/EBPα WT-DNA; II) K2Q-DNA; III) K2Ac_a-DNA; and IV) K2Ac_b-DNA. Hydrogen bonds are shown as dashed lines.

Mentions: On acetylation, lysine side chains are changed in that the acetyl group neutralizes the positive charge. Substitution of lysine with glutamine (neutral side chain) mimics the acetylated form11. To examine whether glutamine substitution was consistent with lysine acetylation and to examine the impact of C/EBPα acetylation on its structure (Fig. 3a), we conducted MD simulations on four model systems. In addition to the WT DNA and acetylation mimic (K2Q-DNA) systems, two acetylated models were also included in this study. The H-N-C=O dihedral angle of the acetylated lysine residues was set to 0° and 180° in the two acetylated models (hereafter termed as K2Ac_a-DNA and K2Ac_b-DNA, respectively, Fig. 3b; see Computational methods section for details). The backbone atoms of the four models showed similar fluctuations during MD simulations (Supplementary Fig. 3). As compared with the C/EBPα WT–DNA complex, lysine acetylation (K2Ac_a-DNA and K2Ac_b-DNA) and acetylation mimic (K2Q-DNA) raised the calculated protein–DNA-binding free energies substantially (Table 1). This indicates a decrease in DNA-binding potency of the models (K2Ac_a-DNA, K2Ac_b-DNA and K2Q-DNA). According to the crystal structure of C/EBPα WT DNA, one of the key interactions is formed between K298 and A−5 (ref. 18). During MD simulations, the three ammonium hydrogen atoms of K302 formed hydrogen bonds with the phosphate oxygen atoms of T−4 (Fig. 3c, Supplementary Table 3). The electrostatic interaction energy between K298 and K302, and DNA residues T−4 and A−5 is a major component of the total protein–DNA-binding energy. Both acetylated lysine and glutamine formed significantly less hydrogen bonds with DNA during the 3 ns MD simulations (Supplementary Table 3). Furthermore, the electrostatic stabilization between the C/EBPα protein and DNA residues were significantly lowered in the mutant systems (Supplementary Table 4). Acetylation might also destabilize the C/EBPα structure. 15 μs MD simulations have shown higher conformational fluctuations and loss of enzymatic activity both during acetylation and mutation of K104 to glutamine in RAS23. Similar structural destabilization might also be responsible for the loss of C/EBPα transcriptional activity on acetylation.


Acetylation of C/EBP α inhibits its granulopoietic function
Molecular dynamics simulation of acetylation at K298 and K302.(a) Diagram of the C/EBPα protein. The positions of the three transactivation elements (TEs), basic region-leucine zipper (BR-LZ) and putative acetylation sites for GCN5 are indicated. (b) The two conformations of the acetylated lysine (ACK) side chain built into the starting coordinates of the acetylated models and preserved during the MD simulations. (c) MD snapshots during 3 ns simulation showing selected protein–DNA interactions: I) C/EBPα WT-DNA; II) K2Q-DNA; III) K2Ac_a-DNA; and IV) K2Ac_b-DNA. Hydrogen bonds are shown as dashed lines.
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f3: Molecular dynamics simulation of acetylation at K298 and K302.(a) Diagram of the C/EBPα protein. The positions of the three transactivation elements (TEs), basic region-leucine zipper (BR-LZ) and putative acetylation sites for GCN5 are indicated. (b) The two conformations of the acetylated lysine (ACK) side chain built into the starting coordinates of the acetylated models and preserved during the MD simulations. (c) MD snapshots during 3 ns simulation showing selected protein–DNA interactions: I) C/EBPα WT-DNA; II) K2Q-DNA; III) K2Ac_a-DNA; and IV) K2Ac_b-DNA. Hydrogen bonds are shown as dashed lines.
Mentions: On acetylation, lysine side chains are changed in that the acetyl group neutralizes the positive charge. Substitution of lysine with glutamine (neutral side chain) mimics the acetylated form11. To examine whether glutamine substitution was consistent with lysine acetylation and to examine the impact of C/EBPα acetylation on its structure (Fig. 3a), we conducted MD simulations on four model systems. In addition to the WT DNA and acetylation mimic (K2Q-DNA) systems, two acetylated models were also included in this study. The H-N-C=O dihedral angle of the acetylated lysine residues was set to 0° and 180° in the two acetylated models (hereafter termed as K2Ac_a-DNA and K2Ac_b-DNA, respectively, Fig. 3b; see Computational methods section for details). The backbone atoms of the four models showed similar fluctuations during MD simulations (Supplementary Fig. 3). As compared with the C/EBPα WT–DNA complex, lysine acetylation (K2Ac_a-DNA and K2Ac_b-DNA) and acetylation mimic (K2Q-DNA) raised the calculated protein–DNA-binding free energies substantially (Table 1). This indicates a decrease in DNA-binding potency of the models (K2Ac_a-DNA, K2Ac_b-DNA and K2Q-DNA). According to the crystal structure of C/EBPα WT DNA, one of the key interactions is formed between K298 and A−5 (ref. 18). During MD simulations, the three ammonium hydrogen atoms of K302 formed hydrogen bonds with the phosphate oxygen atoms of T−4 (Fig. 3c, Supplementary Table 3). The electrostatic interaction energy between K298 and K302, and DNA residues T−4 and A−5 is a major component of the total protein–DNA-binding energy. Both acetylated lysine and glutamine formed significantly less hydrogen bonds with DNA during the 3 ns MD simulations (Supplementary Table 3). Furthermore, the electrostatic stabilization between the C/EBPα protein and DNA residues were significantly lowered in the mutant systems (Supplementary Table 4). Acetylation might also destabilize the C/EBPα structure. 15 μs MD simulations have shown higher conformational fluctuations and loss of enzymatic activity both during acetylation and mutation of K104 to glutamine in RAS23. Similar structural destabilization might also be responsible for the loss of C/EBPα transcriptional activity on acetylation.

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.

No MeSH data available.