【作者】 王涛；

【导师】 李志伟；

【作者基本信息】 吉林大学 ， 生物药学， 2017， 博士

【摘要】 转录因子NF-κB调节数以百计的基因转录,其功能涉及细胞存活、细胞增殖、炎症反应和癌症等。通过统计已报道的κb site(NF-κB binding site,NF-κB结合位点),NF-κB共识序列(consensus sequence)被推断为GGGRNNYYCC(N代表任何碱基,R代表嘌呤,Y代表嘧啶)。多种NF-κB的x射线晶体结构结果显示,p50结合于5’-GGGRN-3’,p65结合于5’-YYCC-3’,这与之前的统计结果一致。研究者通过使用SELEX(systematic evolution of ligands by exponential enrichment)和PBM(protein-binding microarrays)验证了以上结论并对NF-κB可能的结合序列进行了扩展。其中,通过使用PBM,3285个潜在的κB sites与10种NF-κB二聚体的结合能力被系统分析,并相对定量以z-score值反映。在哺乳动物基因组中,DNA Cp G位点的C5甲基化是一种常见的影响基因转录的表观遗传调控方式。基因组中,部分区域的Cp G密度远高于其它区域,这部分区域被称为CGI(Cp G island,Cp G岛)。CGI大多位于基因启动子中,并且CGI很少发生甲基化。转录调控区域的CGI甲基化会明显抑制下游基因的转录。低Cp G密度的转录调控区发生的DNA甲基化对基因转录的影响目前还不是十分清晰,不过一般认为转录因子结合位点或其周围的Cp G发生甲基化会抑制转录因子的结合。然而,单一CGI的甲基化是否会影响基因的转录仍然是一个尚待研究的领域。已报道的与p65:p50二聚体结合的κB site大多符合NF-κB共识序列GGGRNNYYCC,但是反过来,基因组中的NF-κB共识序列却未必能够在NF-κB激活时加强下游基因转录。有时相邻的κb sites中某些可以但另一些不能同NF-κB结合,有时同样的κB site序列在某些位置有功能但在其它位置则无功能,这些发现提示,在NF-κB共识序列之外,还有其它因素(比如:κb sites的周围序列)影响NF-κB的结合或NF-κB结合后的功能。为探索有功能的κb sites周围碱基潜在的规律,我们从已发表的文献和数据库中挑选出人已明确结合位点且完全符合GGGRNNYYCC的κB sites,共计70个。我们分析了这70个κB site上下游各50 bp(base pair)范围内的碱基出现频率,发现κB site-1位置C碱基(-1C)出现频率明显低于其它碱基。我们最初猜测-1Cκb site较少的原因在于-1C可能会影响κb site的功能。因此我们使用报告基因分析,研究了CCL2(C-C motif chemokine ligand 2)κb site-1位置的T/C突变对下游基因的影响。结果显示-1C突变组的luciferase活力较野生型-1T组无明显改变,这提示-1位置T/C突变对κB site的功能无直接影响。NF-κB共识序列的第一个碱基是G,-1位置为C时可形成一处Cp G位点。-1C不影响NF-κB的功能,因此我们推测-1 Cp G(-1C与+1G组成的Cp G位点)的甲基化会抑制NF-κB与κB site的结合。为证明这个推测,我们进行了体外的DAPA(DNA affinity precipitation assay)。我们发现FABP6(fatty acid binding protein 6)κB site和CCL2κb site-1位置为me Cp G(methylated Cp G)时与p65、p50的结合能力较-1位置为Cp G或Ap G时明显降低,但-1位置为Cp G与Ap G则无明显差别。这提示κB site-1C并不影响NF-κB与κB site的结合,但是-1 Cp G的甲基化会抑制NF-κB与κB site的结合。为再次验证以上结论并且分析-1 Cp G的甲基化是否对κB site在细胞内的功能产生影响,我们使用报告基因分析检查了FABP6κB site和CCL2κb site-1Cp G甲基化对κb site功能的影响。我们发现κB site-1位置Wp G/me Cp G(W代表A或T)的突变明显降低了转染细胞受到TNFα(tumor necrosis factorα)处理后luciferase活力的增强倍数,然而κB site-1位置Wp G/Cp G的突变却对此无明显影响。这些结果提示κB site-1 Cp G的甲基化会抑制κB site的功能。通过RT-PCR(reverse transcription-PCR)实验,我们验证了在U937细胞中包含-1Cκb site的基因(-1C基因)也可以被NF-κB激活,提示-1CκB site可能处于低甲基化状态以维持其功能。因此我们通过BSP(bisulfite sequencing PCR)法检查了U937、8226和HEK 293T细胞中所有的六个-1CκB site周围Cp G的甲基化状态。结果显示,除U937细胞中的TFPI2(tissue factor pathway inhibitor 2)κB site和HEK 293T细胞中的IRF7(interferon regulatory factor 7)κB site以外,其它-1CκB site均处于低甲基化状态。这验证了-1CκB site需要处于低甲基化状态的猜测。并且我们发现所有的-1CκB site均位于CGI中,但是-1D(D指代A、T或G)κB site只有22%位于CGI中。这提示只有位于CGI中的-1CκB site才能在进化中保守存在。TFPI2和IRF7的κB site-1位碱基为C且处于CGI中,但BSP分析结果显示,U937细胞中TFPI2和HEK 293T细胞中IRF7的κB site周围甲基化状态很高。RT-PCR结果显示U937细胞中的TFPI2基因和HEK 293T细胞中的IRF7基因不能响应NF-κB的激活。但使用甲基转移酶抑制剂5-Aza-Cd R(5-Aza-2’-deoxycytidine)可恢复这两个基因对NF-κB激活的响应。这再一次证明了甲基化在NF-κB调控下游基因中的关键作用。因此,-1C甲基化提供了一种额外的调节下游基因转录的方式。部分κb site因为其序列的特点,可以与NF-κB有多种结合方式。因为NF-κB不同的结合方式,在我们的统计中有四个κb site-1位置可以为C可以为D。通过BSP实验我们发现-1C or D且不位于CGI中的FLRG(follistatin-related gene)κb site在U937和HEK 293T细胞中处于高甲基化状态。为判断多结合方式的κb site“-1 Cp G”甲基化是否会对NF-κB的结合产生影响,我们选择-1C or D的PTX3(pentraxin 3)κB site和FLRGκB site进行了DAPA实验。结果显示PTX3κb site与FLRGκb site的“-1 Cp G”的甲基化并不会抑制NF-κB的结合。进一步我们合成了排除了“-1C”结合方式的DNA,并以此进行DAPA实验,结果显示这种突变未降低或只在一定程度上降低了NF-κB的结合能力。这提示我们在“-1 Cp G”甲基化时,NF-κB可选择其它-1D的结合方式。DNA甲基化总是在DNA双链中对称出现。为探索-1 Cp G中哪条链的甲基化对NF-κB的结合影响更大,我们使用半甲基化的DNA进行了DAPA实验。结果显示-1C的甲基化显著地抑制了NF-κB与κB site的结合,但反向互补链+1C的甲基化对NF-κB与κB site的结合却几乎没有影响。前面的实验结果表明在大多数情况中,-1Cκb sites只有在CGI中才是有功能的。因此我们通过对多种脊椎动物中VCAM1(vascular cell adhesion molecule1)、CCL2和Rel B基因的启动子和增强中的κb site进行统计分析,来判断潜在的-1Cκb sites在进化中是否保守。我们发现在进化中,位于CGI中的κb site-1位置可以是任何碱基,然而不位于CGI中的κb site-1位置严格控制为非C。这也说明在进化中,-1Cκb site只有位于CGI中才可以保守存在。在本研究中,我们发现κb site的-1 Cp G的甲基化可以影响NF-κB与κb site的结合,从而影响κb site的功能。在κb site的-1 Cp G中,-1C的甲基化明显抑制了NF-κB的结合,而反向互补链+1C的甲基化所产生的作用却不明显。我们也发现对于多结合方式的κb sites,“-1 Cp G”的甲基化对NF-κB的结合无明显影响,这可能是因为“-1 Cp G”甲基化时,NF-κB可选择其它-1D的结合方式。为维持-1Cκb site的功能,在进化中只有位于CGI中的-1Cκb site才是保守的。我们的研究表明与κb site紧临的单个Cp G的甲基化可以为NF-κB下游基因的表达提供一种额外的调控机制。更多还原

【Abstract】 Transcription factor NF-κB regulates expression of hundreds of genes involved in the cell survival,proliferation,inflammation,cancer and other pathophysiological conditions.The classical NF-κB 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-κB binding sites(κB 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-κB dimers to a wide-ranging set of 3,285 potential κB 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-κB activation.This functional difference could occur even between two adjacent κB sites,or two identical κB sites surrounded by different DNA sequences.These findings suggested that other factor(s),such as the DNA sequences surrounding the κB sites,in addition to the consensus sequence itself,might affect either NF-κB binding or its activity after binding to DNA.In order to identify these potential factors in DNA sequences surrounding the κB sites,we compared DNA sequences 50 base pairs(bps)upstream and downstream the κB sites that were experimentally identified as GGGRNNYYCC.We found that the frequency of a cytosine at-1 position of κB 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)κb site on NF-κB 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-κB function.The first base of the NF-κB consensus sequence is a guanine.It will form a Cp G dinucleotide with a-1C nucleotide.Since-1C doesn’t affect NF-κB function,we then asked whenther-1 Cp G(consist of-1C and +1G)methylation would substantially reduce the binding affinity between DNA and NF-κB.We carried out DNA affinity precipitation assays(DAPAs)using κB 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-κB 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-κB binding capacity.To determine whether reduced NF-κB 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 κb site or CCL2 κb site would affect the function of NF-κB in living cells.It was shown that the ratios between luciferase activities in cells treated with or without TNFα 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 κB site function.We then verified that upon TNFα activation of NF-κB in U937 cells,transcription of-1C genes(-1C κB site-containing genes)could be increased by reverse transcription-PCR(RT-PCR),suggesting that the-1C κB 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 κB sites in U937,8226 and HEK 293 T three cell lines by bisulfite sequencing PCR(BSP).It was shown that among all six κB sites examined,only tissue factor pathway inhibitor 2(TFPI2)κB site in U937 cells and interferon regulatory factor 7(IRF7)κB site in HEK 293 T cells were hyper-methylated.This confirmed that-1C κb sites are usually hypo-methylated.We also found that all-1C κB sites are in CGIs whereas only 22% of the-1D κB sites(D = A,G,or T)are in CGIs.These findings suggested that-1C κB sites are evolutionarily conserved only when they locate within CGIs.The-1C κB 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 TNFα activation of NF-κB,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-κB 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 κb sites have mutiple binding patterns because of their sequence specialty.Due to the mutiple NF-κB binding patterns,four κb sites could but do not have to have a cytosine at the-1 position.Among these for κb sites,we found that follistatin-related gene(FLRG)κb 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 κB sites with mutiple NF-κB binding patterns would affect NF-κB binding,we carried out DAPA using oligonucleotides with PTX3(pentraxin 3)or FLRG κB site.It was shown that “-1 Cp G” methylation at the PTX3 or FLRG κB site did not inhibit the NF-κB binding.Further studies indicated that mutation that only abolish “-1C” binding pattern did not or slightly reduce the NF-κB binding.These results suggested that NF-κB 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-κB binding,we carried out DAPA using oligonucleotides with hemimethylated FABP6 κb site.Interestingly,the-1C methylation substantially inhibited NF-κB binding,whereas the methylation of the +1C of reverse complementary strand did not significantly inhibited NF-κB binding.The data shown above suggested that in most cases,-1C κB 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 κB sites with-1C would be evolutionarily conserved.We found that there are barely-1C κb sites that are not located in CGI.These analyses suggested that a-1C κB 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-κB and κB sites,and thereafter pathophysiological function of-1C κB sites.Methylation of-1C(hemimethylation),but not the +1C of reverse complementary strand(hemimethylation),substantially inhibited the NF-κB binding.We also found that “-1 Cp G” methylation of κb sites with mutiple NF-κB binding patterns did not inhibite NF-κB binding,which may be due to that NF-κB could select a-1D binding pattern when “-1 Cp G” is methylated.To maintain the-1C κB sites function,a-1C κB site is evolutionally conserved only when locating in a CGI.Our study suggests that epigenetic modification of the Cp G dinucleotide adjacent to NF-κB consensus sequences would provide an extra regulatory manner to the expression of NF-κB target genes.更多还原