The bZIP mutant CEBPB (V285A) has sequence specific DNA binding propensities similar to CREB1

Ray, Sreejana; Ufot, Aniekanabasi; Assad, Nima; Singh, Jocelyn; Durell, Stewart R.; Porollo, Aleksey; Tillo, Desiree; Vinson, Charles

doi:10.1016/j.bbagrm.2019.02.002

Cited by 4 publications

(6 citation statements)

References 35 publications

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“…Single-stranded DNA 60-mers on the array were double-stranded using T7 DNA polymerase using either cytosine, 5mC (NEB), or 5hmC (Zymo Research) . Enzymatic methylation of CG dinucleotides on PBMs (DNA(5mCG)) and protein binding experiments ,,− were performed as described previously.…”

Section: Methodsmentioning

confidence: 99%

Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182

et al. 2021

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Zta, the Epstein–Barr virus bZIP transcription factor (TF), binds both unmethylated and methylated double-stranded DNA (dsDNA) in a sequence-specific manner. We studied the contribution of a conserved asparagine (N182) to sequence-specific dsDNA binding to four types of dsDNA: (i) dsDNA with cytosine in both strands ((DNA(C|C)), (ii, iii) dsDNA with 5-methylcytosine (5mC, M ) or 5-hydroxymethylcytosine (5hmC, H ) in one strand and cytosine in the second strand ((DNA(5mC|C) and DNA(5hmC|C)), and (iv) dsDNA with methylated cytosine in both strands in all CG dinucleotides ((DNA(5mCG)). We replaced asparagine with five similarly sized amino acids (glutamine (Q), serine (S), threonine (T), isoleucine (I), or valine (V)) and used protein binding microarrays to evaluate sequence-specific dsDNA binding. Zta preferentially binds the pseudo-palindrome TRE (AP1) motif ( T –4 G –3 A -2G / C 0 T 2 C 3 A 4 ). Zta (N182Q) changes binding to A 3 in only one half-site. Zta(N182S) changes binding to G 3 in one or both halves of the motif. Zta(N182S) and Zta(N182Q) have 34- and 17-fold weaker median dsDNA binding, respectively. Zta(N182V) and Zta(N182I) have increased binding to dsDNA(5mC|C). Molecular dynamics simulations rationalize some of these results, identifying hydrogen bonds between glutamine and A 3 , but do not reveal why serine preferentially binds G 3 , suggesting that entropic interactions may mediate this new binding specificity.

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Section: Methodsmentioning

confidence: 99%

Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182

et al. 2021

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“…The design of the Agilent 40K array has been described previously. ,− Specifically, we used the “HK” array design available on the NCBI Gene Expression Omnibus platform GPL11260 (). HK microarrays with 40330 single-stranded DNA 60-mers were double-stranded to generate five types of dsDNA using either cytosine (C, NEB), 5-methylcytosine (5mC, NEB), 5-hydroxymethylcytosine (5hmC, Zymo Research), 5-formylcytosine (5fC, TriLink Biotechnologies), or 5-carboxylcytosine (5caC, TriLink Biotechnologies). ,, The double-stranding efficiency was monitored using either 4% Cy3-dCTP (GE Healthcare) or 4× SYBR-Gold (LifeTechnologies Corp.) DNA intercalating dye. These microarrays were used for protein binding microarray (PBM) experiments in which a GST-tagged bZIP DNA binding domain binds to the DNA on the microarray slide.…”

Section: Experimental Proceduresmentioning

confidence: 99%

“…These microarrays were used for protein binding microarray (PBM) experiments in which a GST-tagged bZIP DNA binding domain binds to the DNA on the microarray slide. Protein binding reactions were performed as described previously. ,,, Briefly, 180 ng of plasmid containing DNA binding domains was used to express proteins using the PureExpress in vitro transcription translation kit (NEB) in a 25 μL reaction volume following the manufacturer’s instructions. The double-stranded arrays and in vitro -expressed proteins were blocked with 4% milk and BSA before the binding reaction.…”

Section: Experimental Proceduresmentioning

confidence: 99%

“…37 At a structural level, this work highlighted an alanine residue in CREB1, cJun, and cFos, a serine residue in Zta, and a valine in CEBPB explaining the altered DNA binding specificity. 37 Mutagenesis experiments that mutated serine 189 in Zta 51 and a conserved valine in CEBPB 52 highlighted the important roles of these residues in determining the specificity of binding to 5mC and 5hmC. In this study, we examined the effects of 5mC, 5hmC, 5fC, and 5caC in one strand of dsDNA on sequence-specific binding of five bZIP domains (CREB1, ATF2, Zta, ATF3|cJun, and cFos| cJun).…”

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bZIP Dimers CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun Prefer to Bind to Some Double-Stranded DNA Sequences Containing 5-Formylcytosine and 5-Carboxylcytosine

et al. 2020

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In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.

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“…23 specific DNA binding ability. 26,27 Here, the contribution of 5mC and 5hmC in one strand of dsDNA to NFATc2 binding was examined.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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NFATc2 is a DNA binding protein in the Rel family transcription factors, which binds a CGGAA motif better when both cytosines in the CG 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, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and a cytosine in the second strand. ATTTCCAC, the complement of the core GGAA motif, is better bound as ssDNA compared to dsDNA. dsDNA containing the 5-mer CGGAA with either 5mC or 5hmC in one DNA strand is bound stronger than CGGAA. In contrast, the reverse complement TTCCG is bound weaker when it contains 5mC. Analysis of the available NFATc2:dsDNA complexes rationalizes these PBM data.

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The bZIP mutant CEBPB (V285A) has sequence specific DNA binding propensities similar to CREB1

Cited by 4 publications

References 35 publications

Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182

Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182

bZIP Dimers CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun Prefer to Bind to Some Double-Stranded DNA Sequences Containing 5-Formylcytosine and 5-Carboxylcytosine

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

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