| Matrix Attachment Regions (MARs) are the DNA sequences with A/T rich nucleotides that may be involved in anchoring DNA/chromatin to the nuclear matrix. The displacement proteins specifically bind to MAR regions in chromatin to facilitate the mediated displacement of H1 histones, and the enrichment of those proteins might open the chromatin structure of TM6 regions. MAR plays an important role in the maintance and modification of the DNA/ chromatin structure and the regulation of the gene expression.TM6 is a matrix attachment region isolated from the genomic DNA of tobacco, which can strongly bind to the nuclear matrix and significantly enhance the transgene expression in transgenic plants of tobacco. To gain insight into the regulatory mechanism of TM6 by which transcription enhancement of transgene occurs, we give the detailed analysis of the expression variation of flanking transgenes drived by different promoters in different expression systems. We present the main results as follows:1. TM6 is a novel element to increase the transgene expression levels in both dicotyledons (tobacco) and monocotyledons (arabidopsis). The difference of the cell development in plants and calli does not influnce the function of TM6. Four different promoters (P35S, PPNZIP, PCOR and Pmini35S) have been used to test the influence of MAR on transgenic plants, and the results show that TM6 does not change the gene expression pattern. The result, that MAR increase gene expression in desired tissues with tissue?specific promoters, is a new way to enhance the transgene expression.2. Assays of transgenic plants show that the expression level of nptâ…¡, the quantitative GUS activity and the transformation efficiency are quite consistent, TM6 transgenic plants have higher transformation efficiency than those without TM6. We presume that TM6 increases the transgene efficiency through the increase expression levels of nptâ…¡, and the TM6 transgenic plants show the stronger seed germination.3. Deletion analysis shows that TM6II (551-1193 bp) plays 79.1% role of the whole TM6 sequence. The effect of the site?specific deletion of one topoisomerase II binding site, one AT?box and one MRS element in TM6II indicate that they perform the vast majority of the enhancement mediated from TM6. There is some functional redundancy in the contribution to open the TM6 chromatin structure and recruit transcription factors, increasing the genes transcription.4.According to the micrococcal nuclease accessibility analysis, the CaMV 35S promoter adjacent to the TM6 is degraded more rapidly than the control without MAR. Considering the increasing accessibility to micrococcal nuclease would result in the decrease of PCR products for the regions of interest, the difference reveals that TM6 plays a role in nucleosome remodeling of the promoter region. The deletion of the four sites determines the effect of TM6 on the micrococcal nuclease accessibility.5. Two potential genes encoding TM6 sequence?binding proteins, NtMBP1 and NtHMGB, are screened from the cDNA pool by yeast one?hybrid method. The two proteins show high affinity with two specific fragments, TM6II?I (761 to 870 bp) and TM6II?II (934 to 1013 bp), by electrophoresis mobility shift assay (EMSA). NtHMGB can specifically bind to MRS element of TM6, and NtMBP1 might associate with other elements on TM6. These potential multiple targets implicate the functional complexity of the TM6.6. Proposed models for the chromatin regulation of MARs on gene expression. We persume that some DNA duplex?destabilized enzymes and chromatin regulated factors, such as the helicases and topoisomerases, bind to specific elements in TM6 loosening the TM6 chromatin. The NtMBP1 and NtHMGB proteins act as the architectural factors to displace the H1 histones to decrease compactness of the chromatin fiber at the TM6 regions. Meanwhile the transcription factors (such as FACTs) bind to the opening chromatin regions to increase the transcription.
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