Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
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
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.
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