title: C/EBPβ (CEBPB) protein binding to the C/EBP|CRE DNA 8-mer TTGC|GTCA is inhibited by 5hmC and enhanced by 5mC, 5fC, and 5caC in the CG dinucleotide.
abstract: During mammalian development, some methylated cytosines (5mC) in CG dinucleotides are iteratively oxidized by TET dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). The effect of these cytosine oxidative products on the sequence-specific DNA binding of transcription factors is being actively investigated. Here, we used the electrophoretic mobility shift assay (EMSA) to examine C/EBPα and C/EBPβ homodimers binding to all 25 chemical forms of a CG dinucleotide for two DNA sequences: the canonical C/EBP 8-mer TTGC|GCAA and the chimeric C/EBP|CRE 8-mer TTGC|GTCA. 5hmC in the CG dinucleotide in the C/EBP|CRE motif 8-mer TGAC|GCAA inhibits binding of C/EBPβ but not C/EBPα. Binding was increased by 5mC, 5fC and 5caC. Circular dichroism monitored thermal denaturations for C/EBPβ bound to the C/EBP|CRE motif confirmed the EMSA. The structural differences between C/EBPα and C/EBPβ that may account for the difference in binding 5hmC in the 8-mer TGAC|GCAA are explored.
authors.affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, United States.
authors.forename: Syed Khund
authors.initials: SK
authors.lastname: Sayeed
authors.affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, United States.
authors.forename: Jianfei
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Room 5120, Building 37, Bethesda, MD 20892, United States.
authors.forename: Bangalore K
authors.initials: BK
authors.lastname: Sathyanarayana
authors.affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, United States.
authors.forename: Jaya Prakash
authors.initials: JP
authors.lastname: Golla
authors.affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, United States. Electronic address: vinsonc@mail.nih.gov.
authors.forename: Charles
authors.initials: C
authors.lastname: Vinson
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism / Q000031:analogs & derivatives; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D003597:Cytosine Nucleotides / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D047108:Embryonic Development / Q000235:genetics; D059372:Nucleotide Motifs / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: C560974:5-carboxylcytosine; C560973:5-formylcytosine; D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C530718:CEBPA protein, mouse; C545013:Cebpb protein, mouse; D003597:Cytosine Nucleotides; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA
keywords: 5hmC, 5fC, 5caC, carboxylation; 5mC; Basic-leucine zipper; C/EBP|CRE motif; C/EBPβ homodimer; CG dinucleotide; DNA binding