Identifying The Characteristics Of UHRF1 Binding DNA Fragments By Using Chromatin Immunoprecipitation Followed By Bisulfite Sequencing Technique In Cancer Cells

DNA methylation is an important epigenetic modification and a reversible genomic DNA molecular marker. Genomic imprinting, gene silencing, X chromosome inactivation, cell reprogramming and tumorigenesis are both regulated dramatically by DNA methylation. UHRF1 is a multiple functions protein that can recognize both DNA methylation and histone modification, and has ubiquitinoylation activity. It has been proved to take part in many biological regulation processes, such as maintenance of DNA methylation, regulation of cell cycles, especially in the tumorigenesis. Previous studies have demonstrated that UHRF1 can not only localize to heterochromatin and stimulate its formation, but also localize to specific euchromatic regions and regulate maintenance of DNA methylation and gene expression. However whether sequence or methylation level of DNA fragment is critical for UHRF1 binding is not clear. In this study, we have established a novel technique based on high-throughput sequencing of bisulfite-treated chromation immunoprecipitated DNA (ChIP-BS-seq), which could directly interrogate protein binding and DNA methylation level in the same DNA molecule in whole genome level. By this technique, we directly discovered the sequence characteristics and DNA methylation status of the chromosome bound by UHRFl and then verified the results by ChIP-qPCR and bisulfite sequencing. The results revealed that UHRF1 binding DNA was mainly located in repetitive sequence region and widely distributed throughout whole genome. The values of Observed/Expected of UHRF1 ChIP Peaks in 3’UTR, exon, promoter and repetitive sequence were 1.7,2.2,1.4 and 1.6, which suggested that UHRF1 preferred to bind to these regions in cancer cells. Among all UHRF1 ChIP Peaks,56.3% were located in gene region. And the percentage of cytosine in ChIPed UHRF1 binding Peaks was 1.01% that was greater than the observed CpG frequency in the whole genome, which demonstrated that UHRF1 preferentially bound to CpG dinucleotide concerntrated region. Because the average DNA methylation level in 3’UTR, exon, promoter of Peaks of UHRF1 ChlP group was above 0.7, UHRF1 was supposed to bind to DNA fragments with high methylation level. Further analysis revealed that compared to control DNA, the methylation pattern of the UHRF1 binding DNA was in accordance with the speculation of UHRF1’s DNA methylation maintenance function. Moreover, UHRF1 ChIPed Peaks had an overlap with H3K9me3 concerntrated sites. In conclusion, by ChIP-BS-seq technique, we discovered that UHRF1 located mainly in repetative sequence in cancer cells, as well as in some other region all over the genome. No obvious binding sequence specificity of UHRF1 was found. However this protein actually bound to DNA fragments with high methylation level, which indicated the abilities of DNA methylation maintenance and restoration of UHRF1. The results we achieved in this study would well support for deeper studies of the function:of UHRF1 in cancer cells. Furthermore, our results would provide more details of basic mechanisms of UHRF1 function.