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

https://doi.org/10.1016/j.bbagrm.2015.03.002Get rights and content

Highlights

  • We examined effect of 5mC, 5hmC, 5fC and 5caC on DNA binding of C/EBPα and C/EBPβ.

  • DNA-binding of C/EBPβ changes more than C/EBPα when 5mC is oxidized in TGAC|GCAA.

  • 5mC, 5fC and 5caC in CG dinucleotide of TGAC|GCAA enhance DNA binding of C/EBPβ.

  • 5hmC in CG dinucleotide of TGAC|GCAA inhibits binding of C/EBPβ but not C/EBPα.

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.

Introduction

In mammals, most of the cytosines in CG dinucleotides are methylated [1]. Recently, the ten eleven translocation (TET) family of dioxygenases were identified [2], [3] that iteratively oxidize 5mC into 5hmC [3], 5fC, and 5caC [4]. 5fC and 5caC can be removed by mammalian thymine DNA glycosylase (TDG) and replaced by cytosine, completing the demethylation of 5mC [5], [6] that occurs during many developmental stages [7], and physiological and pathological conditions [8], [9]. The abundance of the 5mC oxidative products varies in tissues [10] suggesting they are regulated intermediates with potential biological functions.

The effect of 5mC on the DNA binding of some transcription factors (TFs) has been examined. 5mC inhibits the DNA binding of many TFs involved in housekeeping functions like ETS (CCGGAA), SP1 (CCCGCC), and NRF-1 (CGCCTGCG) [11] suggesting a mechanistic link between hypermethylation of CG islands and gene suppression that is observed in some cancers [12], [13]. Alternatively, 5mC can increase DNA binding of TFs [14] resulting in repression [15] or activation of nearby genes [16], [17], [18], [19]. CG methylation improves binding of the C/EBP family of transcription factors [16], [17], [18] that are critical for activation of tissue specific promoters in many tissues during differentiation [20], [21], [22], [23]. Furthermore, the C/EBPβ|ATF4 heterodimer preferentially binds the methylated CGAT|GCAA where the ATF4 basic region is binding the methylated half-site CGAT|GCAA [24].

A potential consequence of 5mC oxidation is to change the sequence-specific binding of transcription factors (TFs) [25], [26], [27], [28]. In the present study, we used EMSA and CD thermal denaturations to examine the DNA binding of C/EBPα and C/EBPβ to 25 double-stranded DNAs (dsDNA) containing all possible combinations of C, 5mC, 5hmC, 5fC, and 5caC in both cytosines of a CG dinucleotide for two DNA sequences, the canonical palindromic C/EBP 8-mer (TTGC|GCAA) [29], [30] and the chimeric C/EBP|CRE 8-mer TTGC|GTCA [31], [32], [33]. C/EBP family members are widely expressed transcription factors that regulate cellular proliferation and differentiation with C/EBPα more involved in terminal differentiation [18], [22], [34], [35], [36], [37]. Here we report that the binding of C/EBPβ changes more than C/EBPα when 5mC is oxidized. The strongest change in binding is for C/EBPβ and the chimeric C/EBP|CRE 8-mer suggesting a potential change in function as 5mC becomes oxidized during development [38], physiology [39], and pathology [40].

Section snippets

DNA oligonucleotides

Twenty single-stranded DNA (ssDNA) cartridge-purified 28-mer oligonucleotides (both sense and anti-sense strands for C, 5mC, 5hmC, 5fC, and 5caC) were purchased from W. M. Keck Oligonucleotide Synthesis Facility, Yale to examine two DNA sequences: the C/EBP consensus motif (TTGC|GCAA) and the chimeric C/EBP|CRE motif (TTGC|GTCA). These oligos were validated by capillary electrophoresis and gave single peaks for each oligo. Five 28-mer DNAs for the C/EBP consensus motif (CTGACCCATATTGC|GCAA

C/EBPβ binding to 25 dsDNAs containing 5 different cytosines in the canonical C/EBP motif TTGC|GCAA

5mC in the CG dinucleotide at the center of the consensus C/EBP motif (TTGC|GCAA) and the chimeric C/EBP|CRE (TTGC|GTCA) increases DNA binding of C/EBPα and C/EBPβ proteins [16], [26]. We extended this analysis and evaluated how the three oxidative products of 5mC affected C/EBPα and C/EBPβ binding to these two DNA 8-mers with a CG dinucleotide at the center of the 8-mer. For each sequence, we designed 10 ssDNA 28-mers with the 8-mer motif in the center. Five ssDNAs have different cytosines (C,

Discussion

The potential for 5mC, 5hmC, 5fC, and 5caC to change the sequence specific DNA binding of transcription factors adds complexity to understanding regulated gene expression [6]. The effect of 5mC on sequence-specific DNA binding has been examined for many transcription factors [24], [14] but how the oxidative products of 5mC change DNA binding is less understood. Here, we show that these modifications have modest effects on C/EBPα binding to the palindromic consensus motif (TTGC|GCAA) and the

Acknowledgments

We thank our lab members for their encouragement and support. This work is supported by the intramural research project of the National Cancer Institute, NIH, Bethesda, USA.

References (57)

  • J.A. Hackett et al.

    Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency

    Stem Cell Rep.

    (2013)
  • V. Rishi et al.

    A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors

    Anal. Biochem.

    (2005)
  • J. Robertson et al.

    The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression

    Biochem. Biophys. Res. Commun.

    (2011)
  • C.X. Song et al.

    Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming

    Cell

    (2013)
  • J. Cadet et al.

    TET enzymatic oxidation of 5-methylcytosine, 5-hydroxymethylcytosine and 5-formylcytosine

    Mutat. Res. Genet. Toxicol. Environ. Mutagen.

    (2014)
  • L. Shen et al.

    Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics

    Cell

    (2013)
  • M. Yu et al.

    Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome

    Cell

    (2012)
  • A.M. Deaton et al.

    CpG islands and the regulation of transcription

    Genes Dev.

    (2011)
  • L.M. Iyer et al.

    Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids

    Cell Cycle

    (2009)
  • M. Tahiliani et al.

    Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1

    Science

    (2009)
  • W.A. Pastor et al.

    TETonic shift: biological roles of TET proteins in DNA demethylation and transcription

    Nat. Rev. Mol. Cell Biol.

    (2013)
  • S. Zhu et al.

    CCAAT/enhancer binding protein-beta is a mediator of keratinocyte survival and skin tumorigenesis involving oncogenic Ras signaling

    Proc. Natl. Acad. Sci. U. S. A.

    (2002)
  • D.P. Ramji et al.

    CCAAT/enhancer-binding proteins: structure, function and regulation

    Biochem. J.

    (2002)
  • Y. Liu et al.

    CCAAT/enhancer-binding proteins and the pathogenesis of retrovirus infection

    Future Microbiol

    (2009)
  • S. Kriaucionis et al.

    The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain

    Science

    (2009)
  • J.M. Rozenberg et al.

    All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase II in multiple tissues

    BMC Genomics

    (2008)
  • S.M. Iguchi-Ariga et al.

    CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation

    Genes Dev.

    (1989)
  • S. Hu et al.

    DNA methylation presents distinct binding sites for human transcription factors

    Elife

    (2013)
  • Cited by (0)

    View full text