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| Status |
Public on Mar 01, 2019 |
| Title |
The bZIP mutant CEBPB(V285A) has DNA binding propensities similar to CREB1 [CEBPMut_5mC_5hmC] |
| Platform organism |
synthetic construct |
| Sample organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
The bZIP homodimers CEBPB and CREB1 bind DNA containing methylated cytosines differently. CREB1 binds stronger to the C/EBP half-site GCAA when the cytosine is methylated. For CEBPB, methylation of the same cytosine does not affect DNA binding. The X-ray structure of CREB1 binding the half site GTCA identifies an alanine in the DNA binding region interacting with the methyl group of T, structurally analogous to the methyl group of methylated C. This alanine is replaced with a valine in CEBPB. To explore the contribution of this amino acid to binding with methylated cytosine of the GCAA half-site, we made the reciprocal mutants CEBPB(V285A) and CREB1(A297V) and used protein binding microarrays (PBM) to examine binding to four types of double-stranded DNA (dsDNA): 1) DNA with cytosine in both strands (DNA(C|C)), 2) DNA with 5-methylcytosine (M) in one strand and cytosine in the second strand (DNA(M|C)), 3) DNA with 5-hydroxymethylcytosine (H) in one strand and cytosine in the second strand (DNA(H|C)), and 4) DNA with both cytosines in all CG dinucleotides contain- ing 5mC (DNA(5mCG)). When binding to DNA(C|C), CEBPB (V285A) preferentially binds the CRE consensus motif (TGACGTCA), similar to CREB1. The reciprocal mutant, CREB1(A297V) binds DNA with some similarity to CEBPB with strongest binding to the methylated PAR site 8-mer TTACGTAA. These data demonstrate that V285 residue inhibits CEBPB binding to methylated cytosine of the GCAA half-site.
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| Overall design |
Protein binding microarray (PBM) experiments were performed for the DNA binding domain of CEBPB, CREB1, and the two mutants CEBPB(V285A) and CREB1(A297V). Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded 44K Agilent microarrays (Berger et al., Nature Biotechnology 2006) in order to determine their binding preferences to sequences containing modified cytosines. We modified the double stranding step of the PBM protocol in which the nucleotide mixture contained 5-methylcytosine or 5-hydroxymethylcytosine. Details of the modified cytosine PBM protocol are described in Khund-Sayeed S et al., Integrative Biology, 2016.
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| Contributor(s) |
Tillo D |
| Citation(s) |
30825655 |
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| Submission date |
Mar 19, 2018 |
| Last update date |
May 31, 2019 |
| Contact name |
Desiree Tillo |
| E-mail(s) |
desiree.tillo@nih.gov
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| Phone |
+1-240-760-7289
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| Organization name |
NIH/NCI
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| Street address |
41 Center Dr, Room D310
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL11260 |
Agilent custom ME and HK design array [8mer] |
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| Samples (21)
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| This SubSeries is part of SuperSeries: |
| GSE112020 |
The bZIP mutant CEBPB(V285A) has sequence specific DNA binding propensities similar to CREB1 |
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| Relations |
| BioProject |
PRJNA439166 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE112018_CEBPMut_5mC_5hmC-Z-score-matrix.txt.gz |
6.8 Mb |
(ftp)(http) |
TXT |
| GSE112018_RAW.tar |
24.2 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data are available on Series record |
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