부산대학교 도서관

Despite the abundance of the templates, both human and rodent SINEs are normally expressed at a very low level. DNA methylation-mediated silencing has been proposed as a possible cause of their transcriptional repression. The effect of DNA methylation and the effect of DNA methylation-dependent methyl-CpG-binding domain proteins (MBD proteins) on SINE transcription were studied here. It was shown that both human and rodent SINEs are bound by MeCP2, MBD1 and MBD2. Both human and rodent SINEs were also shown to be occupied by HDAC1, HDAC2 and a component of SWI/SNF complex, Brahma. Human Alus were also found to be occupied by components of two corepressor complexes, SIN3 and NuRD. Whether MBD proteins repress SINE transcription in a DNA methylation-dependent manner was further investigated using systems with low or near absent DNA methylation and, in the case of MeCP2 protein, by its direct removal. MeCP2 was found to have no repressive effect on B1 and B2 expression. RT-PCR analysis showed no increase in B1 and B2 RNA levels in MeCP2 null mice kidneys. ChIP analysis of Dnmt1n/n p53-/- embryonic fibroblasts, which have less than 5% of the normal DNA methylation level, showed significant reduction in MeCP2 and MBD2 binding, confirming that their presence is DNA methylation-dependant. RT-PCR comparison of Dnmt1+/+ p53-/- and Dnmt1n/n p53-/- cells, however, detected no increase in B1 or B2 RNA levels. This was consistent with results obtained from MeCP2 null mice, where lack of MeCP2 did not result in increased B1 and B2 expression and with a previous study involving human Alus (Yu et al., 2001). MBD2 also does not seem to repress SINE activity, as its release following loss of DNA methylation did not result in increased SINE RNA levels. Strikingly, all human and rodent SINEs studied here were found to be bound by transcription factors TFIIIB and TFIIIC at comparable levels with actively transcribed genes. Some RNA polymerase III was also detected, but at levels significantly lower than on active genes, suggesting a defect in RNA polymerase III loading onto SINEs. This occupancy of the transcriptional complex was comparable in cells with normal levels of DNA methylation and in cells with significantly reduced levels of DNA methylation, suggesting that the occupancy is not affected by methylated DNA or DNA methylation-dependent components of chromatin. Indeed, removal of 50% of histone H1 did not result in increased B1 or B2 expression in this study. The fact that all tested SINEs are occupied by TFIIIB and TFIIIC also brings an unprecedented insight into the number of these transcription factors present in the cell.