6mg3
V285A Mutant of the C-terminal bZIP domain of human C/EBPbeta with 16bp Methylated Oligonucleotide Containing Consensus Recognition SequenceV285A Mutant of the C-terminal bZIP domain of human C/EBPbeta with 16bp Methylated Oligonucleotide Containing Consensus Recognition Sequence
Structural highlights
FunctionCEBPB_HUMAN Important transcriptional activator in the regulation of genes involved in immune and inflammatory responses. Specifically binds to an IL-1 response element in the IL-6 gene. NF-IL6 also binds to regulatory regions of several acute-phase and cytokines genes. It probably plays a role in the regulation of acute-phase reaction, inflammation and hemopoiesis. The consensus recognition site is 5'-T[TG]NNGNAA[TG]-3'. Functions in brown adipose tissue (BAT) differentiation (By similarity). Regulates the transcriptional induction of peroxisome proliferator-activated receptor gamma (PPARG).[1] Publication Abstract from PubMedCCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPbeta binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPbeta DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPbeta. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors. Structural basis for effects of CpA modifications on C/EBPbeta binding of DNA.,Yang J, Horton JR, Wang D, Ren R, Li J, Sun D, Huang Y, Zhang X, Blumenthal RM, Cheng X Nucleic Acids Res. 2018 Dec 19. pii: 5253051. doi: 10.1093/nar/gky1264. PMID:30566668[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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