Mutating Zta (182N) changes sequence specific DNA binding (5mC + 5hmC)
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ABSTRACT:
PROVIDER: PRJNA474676 | ENA |
REPOSITORIES: ENA
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Project description:The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated DNA. To study the contribution of the conserved N182 amino acid to sequence specific Zta DNA binding, we replaced it with five other amino acids: serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V). We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of double-stranded DNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)). With unmethylated DNA, Zta(N182S) binds variants of the consensus TRE (TGA-G/C-TCA) motif, such as TGA-G/C-TGA and TCA-G/C-TGA where C at position 3 is replaced with G in one or both half sites of the motif. Zta(N182S) also binds stronger to DNAs containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing GTAA with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C at position 3 is replaced with A. Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined. Zta(N182T) DNA binding is weaker to all types of DNA examined. Our data highlight that mutation of ZtaN182 with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).
2019-06-04 | GSE115351 | GEO
Project description:The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated DNA. To study the contribution of the conserved N182 amino acid to sequence specific Zta DNA binding, we replaced it with five other amino acids: serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V). We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of double-stranded DNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)). With unmethylated DNA, Zta(N182S) binds variants of the consensus TRE (TGA-G/C-TCA) motif, such as TGA-G/C-TGA and TCA-G/C-TGA where C at position 3 is replaced with G in one or both half sites of the motif. Zta(N182S) also binds stronger to DNAs containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing GTAA with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C at position 3 is replaced with A. Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined. Zta(N182T) DNA binding is weaker to all types of DNA examined. Our data highlight that mutation of ZtaN182 with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).
2019-06-04 | GSE115352 | GEO
Project description:The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated double stranded DNA (dsDNA). To study the contribution to sequence specific dsDNA binding of the conserved asparagine in the DNA binding region (Zta(N182)), we replaced it with five other amino acids: serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V). We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of dsDNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)). With unmethylated DNA, Zta binds the consensus TRE motif, also known as the AP1 motif (T-4G-3A-2G/C0T2C3A4). Zta(N182S) binds the variants andwhere C3 is replaced with G3 in one (T-4G-3A-2G/CT2G3A4) or both T-4C-3A-2G/CT2G3A4 half sites. Zta(N182S) also binds stronger to DNA containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing G0T2A3A4 with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C3 is replaced with A3 only on one half site. Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined. Zta(N182T) DNA binding is weaker to all types of DNA examined. Our data highlights that mutation of Zta(N182) with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).
2020-01-01 | GSE126589 | GEO
Project description:The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated double stranded DNA (dsDNA). To study the contribution to sequence specific dsDNA binding of the conserved asparagine in the DNA binding region (Zta(N182)), we replaced it with five other amino acids: serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V). We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of dsDNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)). With unmethylated DNA, Zta binds the consensus TRE motif, also known as the AP1 motif (T-4G-3A-2G/C0T2C3A4). Zta(N182S) binds the variants andwhere C3 is replaced with G3 in one (T-4G-3A-2G/CT2G3A4) or both T-4C-3A-2G/CT2G3A4 half sites. Zta(N182S) also binds stronger to DNA containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing G0T2A3A4 with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C3 is replaced with A3 only on one half site. Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined. Zta(N182T) DNA binding is weaker to all types of DNA examined. Our data highlights that mutation of Zta(N182) with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).
2020-01-01 | GSE126588 | GEO
Project description:Mutating Zta (182N) changes sequence specific DNA binding (5mCG)
| PRJNA474675 | ENA
Project description:Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGAG/CTCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.
2018-05-14 | GSE108273 | GEO
Project description:Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGAG/CTCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.
2018-05-14 | GSE108272 | GEO
Project description: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.
2020-11-16 | GSE141050 | GEO
Project description:The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the binding of Zta to four kinds of double-stranded DNA: 1) DNA containing cytosine on both strands, 2) DNA with 5-methylcytosine (5mC) on one strand and cytosine on the second strand, 3) DNA with 5-hydroxymethylcytosine (5hmC) on one strand and cytosine on the second strand, and 4) DNA where both cytosines in all CG dinucleotides contain 5mC. We compared the resulting data to PBM data for three other B-ZIP proteins (CREB1 and CEBPB homodimers, and cFos-cJun heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M=5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide GCAA stronger when it contains 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, opening the potential for discovery of new viral and host regulatory programs controlled by EBV.
2017-10-26 | GSE100870 | GEO
Project description:The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the binding of Zta to four kinds of double-stranded DNA: 1) DNA containing cytosine on both strands, 2) DNA with 5-methylcytosine (5mC) on one strand and cytosine on the second strand, 3) DNA with 5-hydroxymethylcytosine (5hmC) on one strand and cytosine on the second strand, and 4) DNA where both cytosines in all CG dinucleotides contain 5mC. We compared the resulting data to PBM data for three other B-ZIP proteins (CREB1 and CEBPB homodimers, and cFos-cJun heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M=5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide GCAA stronger when it contains 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, opening the potential for discovery of new viral and host regulatory programs controlled by EBV.
2017-10-26 | GSE100869 | GEO