REL Domain of NFATc2 Binding to Five Types of DNA Using Protein Binding Microarrays.

NFATc2 is a DNA binding protein in the Rel family transcription factors, which binds a motif better when both cytosines in the dinucleotide are methylated. Using protein binding microarrays (PBMs), we examined the DNA binding of NFATc2 to three additional types of DNA: single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with either 5-methylcytosine (5mC, ) or 5-hydroxymethylcytosine (5hmC, ) in one strand and a cytosine in the second strand. , the complement of the core motif, is better bound as ssDNA compared to dsDNA.

The Epstein-Barr Virus B-ZIP Protein Zta Recognizes Specific DNA Sequences Containing 5-Methylcytosine and 5-Hydroxymethylcytosine.

The Epstein-Barr virus (EBV) B-ZIP transcription factor Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the sequence specific DNA binding of Zta to four kinds of double-stranded DNA (dsDNA): (1) DNA containing cytosine in both strands, (2) DNA with 5-methylcytosine (5mC) in one strand and cytosine in the second strand, (3) DNA with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand, and (4) DNA in which both cytosines in all CG dinucleotides contain 5mC. We compared these data to PBM data for three additional B-ZIP proteins (CREB1 and CEBPB homodimers and cJun|cFos heterodimers).

Impact of cytosine methylation on DNA binding specificities of human transcription factors.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences.

5-Methylcytosine (5mC) and 5-Hydroxymethylcytosine (5hmC) Enhance the DNA Binding of CREB1 to the C/EBP Half-Site Tetranucleotide GCAA.

In human and mouse stem cells and brain, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can occur outside of CG dinucleotides. Using protein binding microarrays (PBMs) containing 60-mer DNA probes, we evaluated the effect of 5mC and 5hmC on one DNA strand on the double-stranded DNA binding of the mouse B-ZIP transcription factors (TFs) CREB1, ATF1, and JUND. 5mC inhibited binding of CREB1 to the canonical CRE half-site |GTCA but enhanced binding to the C/EBP half-site |GCAA.

5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4.

We evaluated DNA binding of the B-HLH family members TCF4 and USF1 using protein binding microarrays (PBMs) containing double-stranded DNA probes with cytosine on both strands or 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) on one DNA strand and cytosine on the second strand. TCF4 preferentially bound the E-box motif (CAN|NTG) with strongest binding to the 8-mer CAG|GTGGT. 5mC uniformly decreases DNA binding of both TCF4 and USF1.

Methylated Cytosines Mutate to Transcription Factor Binding Sites that Drive Tetrapod Evolution.

In mammals, the cytosine in CG dinucleotides is typically methylated producing 5-methylcytosine (5mC), a chemically less stable form of cytosine that can spontaneously deaminate to thymidine resulting in a T•G mismatched base pair. Unlike other eukaryotes that efficiently repair this mismatched base pair back to C•G, in mammals, 5mCG deamination is mutagenic, sometimes producing TG dinucleotides, explaining the depletion of CG dinucleotides in mammalian genomes. It was suggested that new TG dinucleotides generate genetic diversity that may be critical for evolutionary change.

GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif.