Cpg Methylation And Endogenous MiRNA Are Responsible For The Tissue-specific Expression Of Human ?-globin Gene In Transgenic Mice

Human globin gene cluster shows erythroid tissue-specific and developmental stages-specific expression. The transcription of globin genes is controlled by the chromatin structure, methylation status of the coding sequence and the flanking regions, cis-regulatory elements and trans-factors. Locus control region (LCR) is the most important cis-element in regulation of?-globin gene expression. Several models (including looping, tracking, linking, topologic alterations, and modification of proteins associated with chromatin) have been proposed to explain the mechanism of human?-globin tissue-specific expression. All the models, directly or indirectly, implicate the methylation pattern of DNA and dynamic chromatin configuration. We previously generated a transgenic mouse strain, in which the spatial and hematopoietic-specific expression of the transgene (enhanced green fluorescent protein, EGFP) is driven by the human?-globin promoter and under controlled of LCR elements (HS2-HS3). The present study extends the previous work to further investigate the mechanism of tissue-specific expression of the gene.To explore the potential role of DNA methylation in expression of human?-globin, we analyzed the EGFP mRNA expression profiles among tissues of the transgenic mice using real-time RT-PCR. The results showed that the EGFP expression was highly specific in hematopoietic tissues. We then investigated the methylation status of the LCR and promoter regions by using sodium bisulfite genomic sequencing. These researches provided significant clues to elucidate the mechanism of the regulation on tissue-specific expression of genes. The main progresses are as follow:1. Expression patterns of the plasmid HS3-2-?P-EGFP-?E are different among various tissues of the transgenic mice. This hematopoietic-specific transgenic mouse model can be used to investigate the mechanism of regulation of human?-globin gene expression in vivo.2). The methylation status of HS3-2 elements in LCR as well as?-globin promoter with upstream region was analyzed. The results showed that all CpGs of the LCR and promoter region in each hematopoietic tissue revealed a tendency toward a hypomethylation pattern, while there is a hypermethylation pattern in non-hematopoietic tissues. The results also demonstrated that different levels of the expression of EGFP transgene in hematopoietic and non-hematopoietic tissues were tightly correlated with different methylated patterns of the?-globin LCR and promoter.3. Differential expression patterns of Dnmt3a and Dnmt3b, but not Dnmt1, in hematopietic and non-henatopoietic tissues, that corresponding to the EGFP expression. It provided the evidence that the differ expression of Dnmt3a and Dnmt3b may constitute one of the mechanisms of the methylations status at CpG sites in?-globin LCR and promoter regions of various tissues.4) Methylation frequencies at some CpG sites of?-globin LCR and promoter region were more predominant, and the methylation status was biased along the CpG positions. The nearer the transcription start site of the transgene plasmid, the higher the methylation frequencies were. The transcription initiation complex plays a critical role in methylation and demethylation, possibly be interfering with the methylation modification after DNA replication.We also investigate whether the transgene tissue-specific expression may be related with the functions of the endogenous microRNA. The 3'-UTR region of human?-globin gene was searched for complimentarily with the existing human miRNAs from miRBase using RNAhybrid software, and 25 miRNAs were identified to be complementary to 3?-UTR region of human?-globin. The analysis of the expression patterns of 5 miRNAs among tissues showed that miR-34c and miR-379 expression profiles were closely correlated with the target EGFP mRNA as well as Dnmt3a and Dnmt3b transcripts in LCR elements and?-globin promoter. Since the expression of the transgene (EGFP) was tightly linked with the methylation status of the individual tissues, we therefore deduced that the differential expression of the transgene in each tissue may be induced by the endogenous miRNAs, causing the differention of the transcription and methylation modification and then impacting on the gene expression.