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. Author manuscript; available in PMC: 2021 Feb 3.
Published in final edited form as: Biochim Biophys Acta. 2015 Mar 13;1849(6):583–589. doi: 10.1016/j.bbagrm.2015.03.002

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.

Syed Khund Sayeed 1, Jianfei Zhao 1, Bangalore K Sathyanarayana 2, Jaya Prakash Golla 1, Charles Vinson 1,*
PMCID: PMC7857639  NIHMSID: NIHMS672096  PMID: 25779641

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 binding 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.

Keywords: 5mC, 5hmC, 5fC, 5caC, Carboxylation, C/EBP|CRE motif, CG dinucleotide, basic-leucine zipper, DNA binding, C/EBPβ homodimer

1. 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].

2. Material and methods

2.2. 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 chimeric C/EBP|CRE motif (TTGC|GTCA). These oliogs were validated by capillary electrophoresis and gave single peaks for each oligo. Five 28-mer DNAs for the C/EBP consensus motif (CTGACCCATATTGC|GCAATCTGACTGAC) termed sense-strand (a) contained different versions of cytosine in bold (C, 5mC, 5hmC, 5fC, and 5caC). The C/EBP consensus motif is underlined and the center of the dyad is marked. Five 28-mer DNAs termed anti-sense strand (b) (GTCAGTCAGATTGC|GCAATATGGGTCAG) contained different versions of cytosine in bold. The chimeric C/EBP|CRE 28-mer on the sense-strand (a) is CTGACCCATATTGC|GTCATCTGACTGAC. The anti-sense strand was end-labeled with γ−32P ATP (specific activity 5000 Ci/mmol, MP Biomedicals) using T4 polynucleotide kinase (New England Biolabs), and was purified by the ProbeQuant G-50 micro column (GE Healthcare Biosciences). The double-stranded DNA (dsDNA) probes were generated by annealing the labeled anti-sense strand and unlabeled sense strand.

2.3. EMSA:

The DNA binding of C/EBPα and C/EBPβ to 25 probes was analyzed by EMSA by incubating the protein homodimers with 32P-labeled dsDNA (7 pM) in EMSA buffer (150 mM KCl, 12.5 mM K2HPO4-KH2PO4, pH 7.4, 1 mM DTT, 0.25 mM EDTA, 0.5 mg/mL BSA, 10% glycerol, 0.02μg/μL poly dIdC, 10 mM MgCl2) in a final volume of the reaction 20 μL. After 20 minutes of incubation at room temperature, protein DNA complex was separated by Polyacrylamide Gel Electrophoresis (PAGE) and autoradiographed.

2.4. Protein expression and purification:

The DNA binding B-ZIP domains of C/EBPα and C/EBPβ were cloned into E. coli protein expression vectors and purified as described previously [41].

2.5. Circular Dichroism spectroscopy:

CD spectroscopy was performed using a Jasco J-720 spectropolarimeter and thermal denaturation curves were fitted [42].

2.6. Crystal structure of transcription factor C/EBPβ homodimer:

The image of the X-ray structure of the C/EBPβ homodimer bound to DNA (pdb2e42) was generated using the program Chimera http://www.cgl.ucsf.edu/chimera/.

3. Results

3.1. 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, 5mC, 5hmC, 5fC and 5caC) in the CG dinucleotide in sense strand CTGACCCATATTGC|GCAATCTGACTGAC and 5 ssDNAs have different cytosines in the anti-sense strand GTCAGTCAGATTGC|GCAATATGGGTCAG. Sense and anti-sense ssDNAs were mixed to make 25 dsDNAs containing different chemical forms of the CG dinucleotide.

Figure 1A presents an EMSA with 3 nM C/EBPβ homodimer binding to 25 radioactive dsDNAs containing different chemical forms of the CG dinucleotide in the palindromic consensus C/EBP motif 8-mer TTGC|GCAA. In dsDNA a CG dinucleotide contains two cytosines, thus the two cytosines may contain either the same or different modifications. For homotypic CG dinucleotide interactions, compared to C|C, 5mC|5mC, 5fC|5fC, and 5caC|5caC increase binding, whereas 5hmC|5hmC decreases binding. For heterotypic interactions, 5hmC|C and 5caC|C is better bound than C|C. The heterotypic CG dinucleotides appear as a sum of the contribution of each cytosine. We highlight the 10 pairs of DNA probes having heterotypic cytosines that are stereochemically equivalent within the palindromic canonical 8-mer and should have similar binding.

Figure 1: C/EBPβ homodimer binding DNA containing modified cytosines:

Figure 1:

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A) Binding of 3 nM C/EBPβ homodimer to 25 dsDNA containing different cytosine forms in the CG dinucleotide in the canonical C/EBP motif TTGC|GCAA. The five homotypic interactions are indicated by * and the ten heterotypic interactions are numbered 1to10. B) Binding of 10 nM C/EBPβ homodimer to 25 dsDNA containing different cytosine forms in the CG dinucleotide in the chimeric C/EBP|CRE motif TTGC|GTCA. C) EMSA showing a half-log dilution from 100 nM to 3 nM for the C/EBPβ homodimer binding three dsDNA containing the C/EBP motif (TTGC|GCAA), one contains unmodified cytosine, one contains two 5mC, and the last contain two 5hmC. D) EMSA showing a half-log dilution from 100 nM to 3 nM for the C/EBPβ homodimer binding four dsDNA containing the chimeric C/EBP|CRE motif (TTGC|GTCA) at its center. One contains unmodified cytosine, one contains two 5mCs, and the last two dsDNA contain both possible 5hmC and 5mC combinations in the CG dinucleotide

3.2. C/EBPβ binding to 25 dsDNAs containing the chimeric C/EBP|CRE 8-mer TTGC|GTCA:

Next we examined C/EBPβ binding to the chimeric C/EBP|CRE motif TTGC|GTCA. Unlike the canonical palindromic C/EBP motif, the chimeric C/EBP|CRE 8-mer TTGC|GTCA is not palindromic and thus the two cytosines in the CG dinucleotide are stereochemically different and thus pairs of heterotypic cytosine interactions are not equivalent. C/EBP|CRE motif contains two B-ZIP half sites, each with a CG dinucleotide at the center of the dyad. The first half site (TTGC|C) is from the palindromic C/EBP motif (TTGC|GCAA) discussed above. The second half site (C|GTCA) is from the palindromic CRE (TGAC|GTCA) bound by the CREB homodimer and other B-ZIP proteins [43].

Figure 1B presents an EMSA with 10 nM C/EBPβ homodimer binding to all 25 chemical forms of the CG dinucleotide in the C/EBP|CRE chimeric 8-mer TTGC|GTCA. A three-fold increase in protein concentration was needed because of weaker C/EBPβ binding to the chimeric site compared to the canonical C/EBP motif. Cytosine modifications change C/EBPβ binding to the chimeric motif (TTGC|GTCA) more than the C/EBP motif (TTGC|GCAA). 5mC modestly increased binding, 5hmC decreased binding, and both 5fC and 5caC increased binding of C/EBPβ binding to the C/EBP|CRE motif.

Figure 1C presents an EMSA for C/EBPβ homodimer binding using a half-log dilution from 100 nM to 3 nM to three dsDNAs: unmodified DNA, dsDNA with two 5mCs, and dsDNA with two 5hmCs in the CG dinucleotide in the canonical C/EBP motif TTGC|GCAA. 5mC increases binding of C/EBPβ homodimer while 5hmC decreases binding compared to unmodified cytosine. Figure 1D presents an EMSA of the C/EBPβ homodimer, using a half-log dilution from 100 nM to 3 nM, binding to four dsDNAs containing the chimeric C/EBP|CRE motif showing that methylation of the CG dinucleotide increases binding and that oxidation of either cytosine to 5hmC inhibits binding compared to unmodified cytosine.

3.3. C/EBPα binding to 25 dsDNA for two motifs:

3 nM C/EBPα homodimer binding to all 25 CG chemical forms of C/EBP motif 8-mer TTGC|GCAA (Figure 2A) is similar to C/EBPβ (Figure 1A). However, 10 nM C/EBPα and C/EBPβ homodimer binding to all 25 CG chemical forms of the C/EBP|CRE chimeric 8-mer TTGC|GTCA is different with 5hmC inhibiting the binding of C/EBPβ but not C/EBPα (Figure 2B).

Figure 2: C/EBPα homodimer binding DNA containing modified cytosines:

Figure 2:

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A) Binding of 3 nM C/EBPα homodimer to 25 dsDNA containing different cytosine forms in the CG dinucleotide in the canonical C/EBP motif TTGC|GCAA. The five homotypic interactions are indicated by * and the ten heterotypic interactions are numbered 1to10. B) Binding of 10 nM C/EBPα homodimer to 25 dsDNA containing different cytosine forms in the CG dinucleotide in the chimeric C/EBP|CRE motif TTGC|GTCA. C) EMSA showing a half-log dilution from 100 nM to 3 nM for the C/EBPα homodimer binding to two dsDNA containing the canonical C/EBP motif (TTGC|GCAA), one dsDNA contains unmodified cytosine, and the other dsDNA contains two 5caCs.

Figure 2C presents an EMSA for C/EBPα homodimer using a half-log dilution from 100 nM to 3 nM binding to two DNAs, an unmodified DNA and one with two 5caCs in the CG dinucleotide in TTGC|GCAA and shows that 5caC increases binding of C/EBPα homodimer by ~ 4-fold.

3.4. Circular dichroism (CD) monitored thermal stability of C/EBPβ bound to chimeric C/EBP|CRE motif (TTGC|GTCA):

We used thermal stability monitored by CD spectroscopy as an independent method to evaluate C/EBPβ binding to modified C/EBP|CRE motifs. Initially, we determined the thermal stability of 16 dsDNAs 28-mers containing different combination of cytosine and modified cytosine using 245 nm. Similar results to our previous study using an E-Box motif were obtained [25]. Among the homotypic dsDNA, 5mC|5mC, 5hmC|5hmC, and 5fC|5fC modestly increase stability whereas 5caC|5caC decreases stability. dsDNAs with only one 5caC has intermediate stability between C|C and 5caC|5caC suggesting the modifications of the two cytosines in the CG dinucleotide are acting independently (Table 1).

Table1: Thermal stability of DNA and C/EBPβ bound to DNA containing cytosine modifications:

Of the 25 forms of the CG dinucleotide, we analyzed 17 forms. 5 had homotypic interactions (C|C, 5mC|5mC, 5hmC|5hmC, 5fC|5fC, and 5caC|5caC; represented by * in the EMSAs) and the remaining 12 had heterotypic interactions.

28-mer dsDNA stability (245 nm) TGAC|CGAA(b)
C/EBP|CRE: TTGC|GTCA (a) Tm (°C) ± SE C(b) 5mC(b) 5hmC(b) 5fC(b) 5caC(b)
C(a) 69.6±0.39 69.3±0.38 69.9±0.33 67.1±0.35
5mC(a) 68.4±0.40 70.6±0.27 69.9±0.22 70.3±0.31 68.6±0.39
5hmC(a) 68.9±0.27 70.3±0.22 71.1±0.16
5fC(a) 70.7±0.21 70.6±0.19
5caC(a) 68.7±0.37 66.9±0.3
C/EBPβ stability bound to dsDNA (222 nm) TGAC|CGAA(b)
C(a) 60.8±0.11 62.1±0.09 60.5±0.09 62.8±0.09
5mC(a) 61.5±0.11 61.5±0.09 60.8±0.10 62.5±0.09 63.5±0.08
5hmC(a) 59.1±0.15 60.7±0.12 58.5±0.15
5fC(a) 63.0±0.08 62.4±0.09
5caC(a) 62.2±0.10 61.9±0.11
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Next we examined C/EBPβ homodimer thermal denaturation monitored by CD spectroscopy at 222 nm as an independent method to assess binding to the chimeric C/EBP|CRE DNA motif. Figure 3A presents CD spectra at 6° C from 200 nm to 300 nm of three samples 1) dsDNA 28-mer containing the chimeric C/EBP|CRE motif TTGC|GTCA, 2) the C/EBPβ homodimer, and 3) the C/EBPβ homodimer bound to dsDNA 28-mer. An increase in negative ellipticity is observed at 222 nm with the addition of DNA as has been observed previously [41] and interpreted as an increase in α-helical structure as the basic region become helical in the major groove of DNA [44,45].

Figure 3:

Figure 3:

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A) CD spectra from 200 nm to 300 nm at 6°C for C/EBPβ, C/EBPβ homodimer bound to a dsDNA 28-mer and unmodified dsDNA 28-mer containing the C/EBP|CRE chimeric motif TTGC|GTCA. B) Circular dichroism at 222 nm of the thermal denaturation at 2 μM dimer of three samples: C/EBPβ (✳), C/EBPβ homodimer bound to a dsDNA 28-mer containing the C/EBP|CRE chimeric motif TTGC|GTCA (□). Thermal denaturation at 245 nm of 2 μM of dsDNA 28-mer containing the C/EBP|CRE chimeric motif TTGC|GTCA (○).

Figure 3B presents the thermal stability of dsDNA monitored at 245 nm with a Tm of 69.6° C. The C/EBPβ homodimer monitored at 222 nm has a Tm of 61.6° C. With heating, the C/EBPβ homodimer cooperatively losses ellipticity at 222 nm as observed previously [41]. The denaturation is well fit using a two-state model of α-helical dimers becoming un-helical monomers. The addition of dsDNA did not dramatically change the thermal stability of the C/EBPβ homodimer (Tm 60.8° C), unlike other B-ZIP dimers where the addition of DNA increases protein stability [16].

We next examined C/EBPβ homodimer binding to 16 dsDNA. Among the homotypic dsDNA, compared to unmodified cytosine 5mC, 5fC and 5caC modestly increase stability of the protein-DNA complex whereas 5hmC decreases the stability, recapitulating what was observed in EMSA (Table1).

3.4. Molecular modeling of C/EBPβ bound to 5mC dsDNA.

To evaluate the structural underpinnings for the differential binding between C/EBPα and C/EBPβ to 5hmC containing C/EBP|CRE motif, we examined an 1.8 angstrom crystal structure of C/EBPβ bound to a canonical DNA motif (pdb 2e42) and highlight the modified cytosine and the amino acids that are different between C/EBPα and C/EBPβ. The closest is Met 294 which is 14 angstroms from 5mC suggesting that the differences in binding are subtle. We presume that the differences in binding affinities of C/EBPα and C/EBPβ are due to the differences in the amino-acids near the modified cytosine.

4. 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 chimeric C/EBP|CRE motif (TTGC|GTCA). In contrast, the DNA binding of the C/EBPβ homodimer is more sensitive to these changes with 5mC [16,24], 5fC, and 5caC increasing binding while 5hmC decreases binding. Introduction of 5hmC results in widening of the major groove at the site of modification[28] inhibit B-Z transition of DNA [46]. 5hmC has been shown to be repressive for transcription [47,48,49]. Interestingly, presence of 5hmC completely abrogates the binding of BHLH protein MAX, USF and HIF1[28]. Alteration of DNA structures has also been reported for 5fC [50] and 5caC[46]. A proteomic screen for proteins binding cytosine and its different moficications has identified novel proteins that bind to 5fC and 5caC with potential regulatory functions [51].

5fC preferentially accumulates at poised enhancers among other gene regulatory elements in mouse embryonic stem cells [52,53,54]. Potentially, C/EBPβ preferential binding to 5fC and 5caC containing C/EBP|CRE motif is important for the regulatory events that occur as parts of the genome become unmethylated. The decrease in C/EBPβ binding to 5hmC containing C/EBP|CRE motif may be significant as this modification accumulates in tissues, particularly the brain [10] [40] [55]. It is plausible that 5hmC may act as a switch by inhibiting C/EBPβ binding and thus promoting C/EBPα binding during differentiation of brain tissue. Transient expression of C/EBPα and C/EBPβ induce Tet2 expression in mouse B cells and facilitate its nuclear translocation upon induction [56] suggesting that C/EBP family members recruit Tet dioxygenases to 5mC containing motifs resulting in oxidation and changes in C/EBP family binding which is critical for the complex changes that occur with demethylation and gene expression.

The methods exist to answer if 5hmC changes the occupancy of C/EBPα and C/EBPβ binding to the modified C/EBP|CRE motif. By performing ChIP with C/EBPα and C/EBPβ antibodies followed by the determination of the methylation and hydroxymethylation status [57], one can answer if C/EBPα is enriched in 5hmC containing DNA. Additionally one can determine the enrichment of 5fC and 5caC in C/EBPα and C/EBPβ ChIP DNA [54]. Our work supports the notion/hypothesis that cytosine modifications regulate transcription in a sequence and context dependent manner and lays the foundation for further exploring the complex interactions between C/EBP family members and 5mC and its oxidative products.

Figure 4.

Figure 4.

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A) Sequence alignment of C/EBPα and C/EBPβ showing the differences in the amino-acids between C/EBPα and C/EBPβ. B). Molecular model of C/EBPβ homodimer bound to consensus DNA sequence. The residues shown in colored spheres represent amino-acids that are different from C/EBPα. 5hmC in one of the DNA strands is shown in magenta.

Highlights of paper.

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

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

  3. 5mC, 5fC and 5caC in CG dinucleotide of TGAC|GCAA enhance DNA binding of C/EBPβ.

  4. 5hmC in CG dinucleotide of TGAC|GCAA inhibits binding of C/EBPβ but not C/EBPα.

Acknowledgments:

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

Abbreviations

C

Cytosine

B-ZIP

basic-leucine zipper

5mC

5-methylcytosine

5hmC

5-hydroxymethylcytosine

5fC

5-formylcytosine

5caC

5-carboxylcytosine

EMSA

electrophoretic mobility shift assay

dsDNA

double-stranded DNA

ssDNA

single-stranded DNA

Footnotes

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References

  • [1].Deaton AM, Bird A, CpG islands and the regulation of transcription, Genes Dev 25 (2011) 1010–1022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Iyer LM, Tahiliani M, Rao A, Aravind L, Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids, Cell Cycle 8 (2009) 1698–1710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A, Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1, Science 324 (2009) 930–935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Cadet J, Wagner JR, TET enzymatic oxidation of 5-methylcytosine, 5-hydroxymethylcytosine and 5-formylcytosine, Mutat Res (2013). [DOI] [PubMed] [Google Scholar]
  • [5].Dawlaty MM, Ganz K, Powell BE, Hu YC, Markoulaki S, Cheng AW, Gao Q, Kim J, Choi SW, Page DC, Jaenisch R, Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development, Cell Stem Cell 9 (2011) 166–175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Pastor WA, Aravind L, Rao A, TETonic shift: biological roles of TET proteins in DNA demethylation and transcription, Nat Rev Mol Cell Biol 14 (2013) 341–356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Zhu S, Yoon K, Sterneck E, Johnson PF, Smart RC, 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 99 (2002) 207–212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Ramji DP, Foka P, CCAAT/enhancer-binding proteins: structure, function and regulation, Biochem J 365 (2002) 561–575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Liu Y, Nonnemacher MR, Wigdahl B, CCAAT/enhancer-binding proteins and the pathogenesis of retrovirus infection, Future Microbiol 4 (2009) 299–321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Kriaucionis S, Heintz N, The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain, Science 324 (2009) 929–930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Rozenberg JM, Shlyakhtenko A, Glass K, Rishi V, Myakishev MV, FitzGerald PC, Vinson C, All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase II in multiple tissues, BMC Genomics 9 (2008) 67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Iguchi-Ariga SM, Schaffner W, CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation, Genes Dev 3 (1989) 612–619. [DOI] [PubMed] [Google Scholar]
  • [13].Tate PH, Bird AP, Effects of DNA methylation on DNA-binding proteins and gene expression, Curr Opin Genet Dev 3 (1993) 226–231. [DOI] [PubMed] [Google Scholar]
  • [14].Hu S, Wan J, Su Y, Song Q, Zeng Y, Nguyen HN, Shin J, Cox E, Rho HS, Woodard C, Xia S, Liu S, Lyu H, Ming GL, Wade H, Song H, Qian J, Zhu H, DNA methylation presents distinct binding sites for human transcription factors, Elife 2 (2013) e00726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Quenneville S, Verde G, Corsinotti A, Kapopoulou A, Jakobsson J, Offner S, Baglivo I, Pedone PV, Grimaldi G, Riccio A, Trono D, In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions, Mol Cell 44 (2011) 361–372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Rishi V, Bhattacharya P, Chatterjee R, Rozenberg J, Zhao J, Glass K, Fitzgerald P, Vinson C, CpG methylation of half-CRE sequences creates C/EBPalpha binding sites that activate some tissue-specific genes, Proc Natl Acad Sci U S A 107 (2010) 20311–20316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Flower K, Thomas D, Heather J, Ramasubramanyan S, Jones S, Sinclair AJ, Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor, PLoS One 6 (2011) e25922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Vinson C, Chatterjee R, CG methylation, Epigenomics 4 (2012) 655–663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Gustems M, Woellmer A, Rothbauer U, Eck SH, Wieland T, Lutter D, Hammerschmidt W, c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs, Nucleic Acids Res 42 (2014) 3059–3072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Buck M, Poli V, Hunter T, Chojkier M, C/EBPbeta phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival, Mol Cell 8 (2001) 807–816. [DOI] [PubMed] [Google Scholar]
  • [21].Darlington GJ, Ross SE, MacDougald OA, The role of C/EBP genes in adipocyte differentiation, J Biol Chem 273 (1998) 30057–30060. [DOI] [PubMed] [Google Scholar]
  • [22].Smink JJ, Leutz A, Rapamycin and the transcription factor C/EBPbeta as a switch in osteoclast differentiation: implications for lytic bone diseases, J Mol Med (Berl) 88 (2010) 227–233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Tanaka T, Yoshida N, Kishimoto T, Akira S, Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene, EMBO J 16 (1997) 7432–7443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Mann IK, Chatterjee R, Zhao J, He X, Weirauch MT, Hughes TR, Vinson C, CG methylated microarrays identify a novel methylated sequence bound by the CEBPB|ATF4 heterodimer that is active in vivo, Genome Res 23 (2013) 988–997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Golla JP, Zhao J, Mann IK, Sayeed SK, Mandal A, Rose RB, Vinson C, Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG|GTG) increases binding of the Tcf3|Ascl1 helix-loop-helix heterodimer 10-fold, Biochem Biophys Res Commun (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Chatterjee R, Vinson C, CpG methylation recruits sequence specific transcription factors essential for tissue specific gene expression, Biochim Biophys Acta 1819 (2012) 763–770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Spruijt CG, Gnerlich F, Smits AH, Pfaffeneder T, Jansen PW, Bauer C, Munzel M, Wagner M, Muller M, Khan F, Eberl HC, Mensinga A, Brinkman AB, Lephikov K, Muller U, Walter J, Boelens R, van Ingen H, Leonhardt H, Carell T, Vermeulen M, Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives, Cell 152 (2013) 1146–1159. [DOI] [PubMed] [Google Scholar]
  • [28].Lercher L, McDonough MA, El-Sagheer AH, Thalhammer A, Kriaucionis S, Brown T, Schofield CJ, Structural insights into how 5-hydroxymethylation influences transcription factor binding, Chem Commun (Camb) 50 (2014) 1794–1796. [DOI] [PubMed] [Google Scholar]
  • [29].Johnson PF, Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference, Mol Cell Biol 13 (1993) 6919–6930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].D. I, An integrated encyclopedia of DNA elements in the human genome, Nature 489 (2012) 57–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Vinson CR, Hai T, Boyd SM, Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design, Genes Dev 7 (1993) 1047–1058. [DOI] [PubMed] [Google Scholar]
  • [32].Vallejo M, Ron D, Miller CP, Habener JF, C/ATF, a member of the activating transcription factor family of DNA-binding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response elements, Proc Natl Acad Sci U S A 90 (1993) 4679–4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].FitzGerald PC, Shlyakhtenko A, Mir AA, Vinson C, Clustering of DNA sequences in human promoters, Genome Res 14 (2004) 1562–1574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Hata K, Nishimura R, Ueda M, Ikeda F, Matsubara T, Ichida F, Hisada K, Nokubi T, Yamaguchi A, Yoneda T, A CCAAT/enhancer binding protein beta isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes, Mol Cell Biol 25 (2005) 1971–1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Nerlov C, The C/EBP family of transcription factors: a paradigm for interaction between gene expression and proliferation control, Trends Cell Biol 17 (2007) 318–324. [DOI] [PubMed] [Google Scholar]
  • [36].Hirata M, Kugimiya F, Fukai A, Ohba S, Kawamura N, Ogasawara T, Kawasaki Y, Saito T, Yano F, Ikeda T, Nakamura K, Chung UI, Kawaguchi H, C/EBPbeta Promotes transition from proliferation to hypertrophic differentiation of chondrocytes through transactivation of p57, PLoS One 4 (2009) e4543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Stoilova B, Kowenz-Leutz E, Scheller M, Leutz A, Lymphoid to myeloid cell trans-differentiation is determined by C/EBPbeta structure and post-translational modifications, PLoS One 8 (2013) e65169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Tsagaratou A, Aijo T, Lio CW, Yue X, Huang Y, Jacobsen SE, Lahdesmaki H, Rao A, Dissecting the dynamic changes of 5-hydroxymethylcytosine in T-cell development and differentiation, Proc Natl Acad Sci U S A (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Hackett JA, Dietmann S, Murakami K, Down TA, Leitch HG, Surani MA, Synergistic Mechanisms of DNA Demethylation during Transition to Ground-State Pluripotency, Stem Cell Reports 1 (2013) 518–531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Pfeifer GP, Xiong W, Hahn MA, Jin SG, The role of 5-hydroxymethylcytosine in human cancer, Cell Tissue Res 356 (2014) 631–641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Rishi V, Potter T, Laudeman J, Reinhart R, Silvers T, Selby M, Stevenson T, Krosky P, Stephen AG, Acharya A, Moll J, Oh WJ, Scudiero D, Shoemaker RH, Vinson C, A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors, Anal Biochem 340 (2005) 259–271. [DOI] [PubMed] [Google Scholar]
  • [42].Krylov D, Mikhailenko I, Vinson C, A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions, EMBO J 13 (1994) 2849–2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Vinson C, Myakishev M, Acharya A, Mir AA, Moll JR, Bonovich M, Classification of Human B-ZIP Proteins Based on Dimerization Properties, Mol Cell Biol 22 (2002) 6321–6335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Vinson CR, LaMarco KL, Johnson PF, Landschulz WH, McKnight SL, In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage, Genes Dev 2 (1988) 801–806. [DOI] [PubMed] [Google Scholar]
  • [45].Shuman JD, Vinson CR, McKnight SL, Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP, Science 249 (1990) 771–774. [DOI] [PubMed] [Google Scholar]
  • [46].Wang S, Long Y, Wang J, Ge Y, Guo P, Liu Y, Tian T, Zhou X, Systematic Investigations of Different Cytosine Modifications on CpG Dinucleotide Sequences: The Effects on the B-Z Transition, Journal of the American Chemical Society 136 (2013) 56–59. [DOI] [PubMed] [Google Scholar]
  • [47].Robertson J, Robertson AB, Klungland A, The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression, Biochem Biophys Res Commun 411 (2011) 40–43. [DOI] [PubMed] [Google Scholar]
  • [48].Choi I, Kim R, Lim HW, Kaestner KH, Won KJ, 5-hydroxymethylcytosine represses the activity of enhancers in embryonic stem cells: a new epigenetic signature for gene regulation, BMC Genomics 15 (2014) 670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Williams K, Christensen J, Pedersen MT, Johansen JV, Cloos PA, Rappsilber J, Helin K, TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity, Nature 473 (2011) 343–348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Raiber EA, Murat P, Chirgadze DY, Beraldi D, Luisi BF, Balasubramanian S, 5-Formylcytosine alters the structure of the DNA double helix, Nat Struct Mol Biol 22 (2015) 44–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Iurlaro M, Ficz G, Oxley D, Raiber EA, Bachman M, Booth MJ, Andrews S, Balasubramanian S, Reik W, A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation, Genome Biol 14 (2013) R119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Song CX, Szulwach KE, Dai Q, Fu Y, Mao SQ, Lin L, Street C, Li Y, Poidevin M, Wu H, Gao J, Liu P, Li L, Xu GL, Jin P, He C, Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming, Cell 153 (2013) 678–691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [53].Cadet J, Wagner JR, TET enzymatic oxidation of 5-methylcytosine, 5-hydroxymethylcytosine and 5-formylcytosine, Mutat Res Genet Toxicol Environ Mutagen 764–765 (2014) 18–35. [DOI] [PubMed] [Google Scholar]
  • [54].Shen L, Wu H, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y, Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics, Cell 153 (2013) 692–706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, Xie ZG, Shi L, He X, Jin SG, Iqbal K, Shi YG, Deng Z, Szabo PE, Pfeifer GP, Li J, Xu GL, The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes, Nature 477 (2011) 606–610. [DOI] [PubMed] [Google Scholar]
  • [56].Di Stefano B, Sardina JL, van Oevelen C, Collombet S, Kallin EM, Vicent GP, Lu J, Thieffry D, Beato M, Graf T, C/EBPalpha poises B cells for rapid reprogramming into induced pluripotent stem cells, Nature 506 (2014) 235–239. [DOI] [PubMed] [Google Scholar]
  • [57].Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li X, Dai Q, Shen Y, Park B, Min JH, Jin P, Ren B, He C, Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome, Cell 149 (2012) 1368–1380. [DOI] [PMC free article] [PubMed] [Google Scholar]

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