The CpG Dinucleotide Adjacent To A ?b Site Affects NF-?B Binding And Function Through Its Methylation

Transcription factor NF-?B regulates expression of hundreds of genes involved in the cell survival,proliferation,inflammation,cancer and other pathophysiological conditions.The classical NF-?B consensus sequence,i.e.the DNA sequences bind to p65/p50 heterodimers,was originally identified as GGGRNNYYCC(where N is any base,R is purine,and Y is pyrimidine)by statistical analysis of the known NF-?B binding sites(?B sites).The studies on the crystal structures of p65 and/or p50 provided further evidence supporting the consensus sequence,and identified 5'-GGGRN-3' and 5'-YYCC-3' as the binding sites for p50 and p65,respectively.Researches with other technologies such as System Evolution of Ligands by Exponential Enrichment(SELEX)and Protein-Binding Microarray(PBM)have confirmed the above finding and extended our knowledge in the consensus sequences.By PBM,the DNA-binding preferences of ten NF-?B dimers to a wide-ranging set of 3,285 potential ?B site sequences were examined systematically,and the DNA-binding preferences were quantitated and transformed into z-scores.In mammalian cells,the C5 methylation at Cp G sites is a significant contributor in the epigenetic regulation of gene transcription.In genome,Cp G dinucleotides in clusters are called Cp G islands(CGIs).CGIs predominantly overlap with gene promoter regions and are typically non-methylated.Methylation of CGIs robustly represses the gene transcription.It is still unclear whether DNA methylation in Cp G-poor regulatory regions will affect gene transcription,although it is believed that DNA methylation around transcription binding sites or within CGIs in the promoter region will prevent the binding of transcription factors.However,the role of a single Cp G site methylation in gene transcription is still a frontier research field.Although majority of p65/p50 binding sequences match the consensus sequence GGGRNNYYCC,the consensus sequences in genome may not be always functional upon NF-?B activation.This functional difference could occur even between two adjacent ?B sites,or two identical ?B sites surrounded by different DNA sequences.These findings suggested that other factor(s),such as the DNA sequences surrounding the ?B sites,in addition to the consensus sequence itself,might affect either NF-?B binding or its activity after binding to DNA.In order to identify these potential factors in DNA sequences surrounding the ?B sites,we compared DNA sequences 50 base pairs(bps)upstream and downstream the ?B sites that were experimentally identified as GGGRNNYYCC.We found that the frequency of a cytosine at-1 position of ?B site(-1C)is substantially lower than that of any of other three nucleotides.At first we wonder whether the reduced-1C frequency might implicate any biological consequence.We therefore used reporter gene assay to study the influence of T/C mutation at-1 position of the C-C motif chemokine ligand 2(CCL2)?b site on NF-?B function.It was shown that there was no significant difference in luciferase activity of transfected cells between-1T(wild type)and-1C(mutation),suggesting that the-1 T/C mutation has no impact on NF-?B function.The first base of the NF-?B consensus sequence is a guanine.It will form a Cp G dinucleotide with a-1C nucleotide.Since-1C doesn't affect NF-?B function,we then asked whenther-1 Cp G(consist of-1C and +1G)methylation would substantially reduce the binding affinity between DNA and NF-?B.We carried out DNA affinity precipitation assays(DAPAs)using ?B sites of fatty acid binding protein 6(FABP6)gene and CCL2 gene.We found that the amount of p65 and p50 binding to the oligonucleotides was dramatically reduced when the oligonucleotides has a methylated Cp G(me Cp G),other than an Ap G or a Cp G,at the-1 position.The NF-?B binding capacity was not significantly affected between-1 Ap G and-1 Cp G oligonucleotides.This finding suggested that it was methylation,not nucleotide replacement itself that caused the decreased NF-?B binding capacity.To determine whether reduced NF-?B binding of-1 Cp G methylation would affect its activity in cells,we used reporter gene assay to determine whether the methylation of-1 Cp G of FABP6 ?b site or CCL2 ?b site would affect the function of NF-?B in living cells.It was shown that the ratios between luciferase activities in cells treated with or without TNF? were not affected by the-1 Wp G/Cp G(W is A or T)mutation,but were significantly suppressed by the single-1 Wp G/me Cp G mutation.These assays suggested that methylation of-1 Cp G,not mutation of the nucleotide itself,could inhibit the ?B site function.We then verified that upon TNF? activation of NF-?B in U937 cells,transcription of-1C genes(-1C ?B site-containing genes)could be increased by reverse transcription-PCR(RT-PCR),suggesting that the-1C ?B sites are hypo-methylated in their promoters/enhancers in order to maintain their functions.To directly detect methylation status of-1C genes,we studied Cp G dinucleotide methylation of all six-1C ?B sites in U937,8226 and HEK 293 T three cell lines by bisulfite sequencing PCR(BSP).It was shown that among all six ?B sites examined,only tissue factor pathway inhibitor 2(TFPI2)?B site in U937 cells and interferon regulatory factor 7(IRF7)?B site in HEK 293 T cells were hyper-methylated.This confirmed that-1C ?b sites are usually hypo-methylated.We also found that all-1C ?B sites are in CGIs whereas only 22% of the-1D ?B sites(D = A,G,or T)are in CGIs.These findings suggested that-1C ?B sites are evolutionarily conserved only when they locate within CGIs.The-1C ?B sites of TFPI2 gene in U937 cells and IRF7 gene in HEK 293 T cells were hyper-methylated,although they are all located in CGI.RT-PCR results shown that upon TNF? activation of NF-?B,there were no significant increases in TFPI2 and IRF7 transcripts in U937 and HEK 293 T cells,respectively.De-methylation with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine(5-Aza-Cd R)restored their response to NF-?B activation substantially.Once more,these results suggested that Cp G methylation is a critical factor regulating gene expression.Therefore,-1C provided an extra target in mediating gene expression.Parts of ?b sites have mutiple binding patterns because of their sequence specialty.Due to the mutiple NF-?B binding patterns,four ?b sites could but do not have to have a cytosine at the-1 position.Among these for ?b sites,we found that follistatin-related gene(FLRG)?b site,which is not located in CGI,were hper-methylated in U937 and HEK 293 T cells by BSP.To determine whether the “-1 Cp G” methylation at the ?B sites with mutiple NF-?B binding patterns would affect NF-?B binding,we carried out DAPA using oligonucleotides with PTX3(pentraxin 3)or FLRG ?B site.It was shown that “-1 Cp G” methylation at the PTX3 or FLRG ?B site did not inhibit the NF-?B binding.Further studies indicated that mutation that only abolish “-1C” binding pattern did not or slightly reduce the NF-?B binding.These results suggested that NF-?B could bind to other patterns when the “-1 Cp G” was methylated.In mammals,DNA methylation always occurs symmetrically at the Cp G dinucleotide.To compare the effect of cytosine methylation of each of two DNA strands on NF-?B binding,we carried out DAPA using oligonucleotides with hemimethylated FABP6 ?b site.Interestingly,the-1C methylation substantially inhibited NF-?B binding,whereas the methylation of the +1C of reverse complementary strand did not significantly inhibited NF-?B binding.The data shown above suggested that in most cases,-1C ?B sites are functional only when locating in CGIs.We therefore analyzed the vascular cell adhesion molecule 1(VCAM1),CCL2 and Rel B gene promoter and enhancer regions in several vertebrates to determine whether potential ?B sites with-1C would be evolutionarily conserved.We found that there are barely-1C ?b sites that are not located in CGI.These analyses suggested that a-1C ?B site was evolutionarily conserved only when it was in a CGI.In this study,we demonstrated that-1 Cp G methylation could affect the binding affinity between NF-?B and ?B sites,and thereafter pathophysiological function of-1C ?B sites.Methylation of-1C(hemimethylation),but not the +1C of reverse complementary strand(hemimethylation),substantially inhibited the NF-?B binding.We also found that “-1 Cp G” methylation of ?b sites with mutiple NF-?B binding patterns did not inhibite NF-?B binding,which may be due to that NF-?B could select a-1D binding pattern when “-1 Cp G” is methylated.To maintain the-1C ?B sites function,a-1C ?B site is evolutionally conserved only when locating in a CGI.Our study suggests that epigenetic modification of the Cp G dinucleotide adjacent to NF-?B consensus sequences would provide an extra regulatory manner to the expression of NF-?B target genes.