The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene

In eukaryotic cells, chromatin is organized as domains or loops that are generated by periodic attachment of the chromatin fiber to protein components of a nuclear matrix, or scaffold. These chromosomal loops may have a function in gene regulation. The length of the chromatin domain encompassing the human apolipoprotein B gene was studied by determining the locations of nuclear matrix attachment sites as well as the boundaries of the DNase I-sensitive domain in cells that express the gene (such as HepG2 and CaCo-2 cells) and in those that do not (HeLa cells). Three nuclear matrix attachment regions (MARs) of the human apolipoprotein B gene have been localized: a 3' -proximal MAR, between nucleotides +43,186 and +43,850; a 5' -proximal MAR, between nucleotides -2,765 and -1,801; and a 5' -distal MAR, between nucleotides -5,262 and -4,048. Both the 3' -proximal and the 5' -distal MARS were present in cells that express the gene (HepG2 and CaCo-2 cells) as well as in cells that do not (HeLa cells), whereas the 5' -proximal MAR was detected only in HepG2 cells. These MARs were located at the bases of chromosomal loops in histone-extracted nuclei in all three cell lines. Various classes of A/T-rich sequences resembling the recognition site for topoisomerase II were present within the MAR-containing fragments. The boundaries of the DNase I-sensitive domain coincide with the positions of the 3' -proximal and 5' -distal matrix attachment sites. These results suggest the existence of a 47.5-kilobase domain that represents a topologically sequestered functional unit containing the coding region and all known cis-acting regulatory elements of the human apolipoprotein B gene.     

Expression of the K-fgf proto-oncogene is controlled by 3'' regulatory elements which are specific for embryonal carcinoma cells.

Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.     

The 5' boundary of the human apolipoprotein B chromatin domain in intestinal cells

The 5' boundary of the chromosomal domain of the human apolipoprotein B (apoB) gene in intestinal cells has been localized and characterized. It is composed of two kinds of boundary elements; the first, functional boundary is an insulator activity exhibited by a 1.8 kb DNA fragment located between -58 and -56 kb upstream of the human apoB promoter. In this region, an enhancer-blocking activity has been mapped to a CTCF binding site that is located upstream of two apoB intestinal enhancers (IEs), the 315 IE and the 485 IE. The CTCF site represents a boundary between two types of chromatin structure: an open, DNaseI-sensitive region 3' of the CTCF site containing the intestinal regulatory elements and a closed, DNaseI-resistant region 5' of the CTCF site. The 1.8 kb fragment harboring the CTCF site also insulated mini-white transgenes against position effects in Drosophila melanogaster. The second, structural boundary is represented by a nuclear matrix attachment region (MAR), situated about 3 kb 5' of the CTCF site. This MAR may represent the 5' anchorage site for a chromosomal loop that functions to bring the intestinal regulatory elements closer to the apoB promoter.     

The human growth hormone locus control region mediates long-distance transcriptional activation independent of nuclear matrix attachment regions

Expression of the human growth hormone (hGH-N) transgene in the mouse pituitary is dependent on a multicomponent locus control region (LCR). The primary determinant of hGH LCR function maps to the pituitary-specific DNase I hypersensitive sites (HS) HSI,II, located 15 kb 5' to the hGH-N gene. The mechanism by which HSI,II mediates long-distance activation of the hGH locus remains undefined. Matrix attachment regions (MARs) comprise a set of AT-rich DNA elements postulated to interact with the nuclear scaffold and to mediate long-distance interactions between LCR elements and their target promoters. Consistent with this model, sequence analysis strongly predicted a MAR determinant in close proximity to HSI,II. Surprisingly, cell-based analysis of nuclear scaffolds failed to confirm a MAR at this site, and extensive mapping demonstrated that the entire 87 kb region encompassing the hGH LCR and contiguous hGH gene cluster was devoid of MAR activity. Homology searches revealed that the predicted MAR reflected the recent insertion of a LINE 3'-UTR segment adjacent to HSI,II. These data point out discordance between sequence-based MAR predictions and in vivo MAR function and predict a novel MAR-independent mechanism for long-distance activation of hGH-N gene expression.     

核基质结合区对转基因表达的影响及其作用机制

核基质结合区(matrix attachment region,MAR)是一段在体外能与核基质结合的富含AT的DNA序列。研究发现MAR能使染色质形成环状结构;将其连到目的基因二侧构建载体并转至生物体中,发现它能增强基因转录表达水平及稳定性,在一定程度上降低转基因个体或细胞系之间转基因的表达水平的差异,这很可能是减低了基因沉默所致。现对MAR的序列特征、MAR对转基因表达的影响及对转基因效应的影响机制进行综述。     

Matrix association region/scaffold attachment region (MAR/SAR) sequence: its vital role in mediating chromosome breakages in nasopharyngeal epithelial cells via oxidative stress-induced apoptosis

Background

Oxidative stress is known to be involved in most of the aetiological factors of nasopharyngeal carcinoma (NPC). Cells that are under oxidative stress may undergo apoptosis. We have previously demonstrated that oxidative stress-induced apoptosis could be a potential mechanism mediating chromosome breakages in nasopharyngeal epithelial cells. Additionally, caspase-activated DNase (CAD) may be the vital player in mediating the chromosomal breakages during oxidative stress-induced apoptosis. Chromosomal breakage occurs during apoptosis and chromosome rearrangement. Chromosomal breakages tend to cluster in certain regions, such as matrix association region/scaffold attachment region (MAR/SAR). We hypothesised that oxidative stress-induced apoptosis may result in chromosome breaks preferentially at the MAR/SAR sites. The AF9 gene at 9p22 was targeted in this study because 9p22 is a deletion site commonly found in NPC.

Results

By using MAR/SAR recognition signature (MRS), potential MAR/SAR sites were predicted in the AF9 gene. The predicted MAR/SAR sites precisely match to the experimentally determined MAR/SARs. Hydrogen peroxide (H₂O₂) was used to induce apoptosis in normal nasopharyngeal epithelial cells (NP69) and NPC cells (HK1). Nested inverse polymerase chain reaction was employed to identify the AF9 gene cleavages. In the SAR region, the gene cleavage frequency of H₂O₂-treated cells was significantly higher than that of the non-treated cells. A few chromosomal breakages were detected within the AF9 region which was previously found to be involved in the mixed lineage leukaemia (MLL)-AF9 translocation in an acute lymphoblastic leukaemia patient. As for the non-SAR region, no significant difference in the gene cleavage frequency was found between the untreated control and H₂O₂-treated cells. Furthermore, H₂O₂-induced cleavages within the SAR region were reduced by caspase-3 inhibitor, which indirectly inhibits CAD.

Conclusions

These results reaffirm our previous findings that oxidative stress-induced apoptosis could be one of the potential mechanisms underlying chromosome breakages in nasopharyngeal epithelial cells. MAR/SAR may play a vital role in defining the location of chromosomal breakages mediated by oxidative stress-induced apoptosis, where CAD is the major nuclease.

     

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the -glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, -glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.     

Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region

Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5' MAR had the effect of localizing beta-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5' MAR is discussed.     

The Influences of Two Plant Nuclear Matrix Attachment Regions (MARs) on Gene Expression in Transgenic Plants

Nuclear matrix attachment regions (MARs) are thought to influencegene expression by anchoring active chromatin to the proteinaceousnuclear matrix. In this study, two plant DNA fragments withstrong MAR activity were selected and tested for their effectson expression of a linked reporter gene in transgenic tobacco.One MAR was isolated from the 5' flanking region of a pea vicilingene previously reported to be expressed in a copy number-dependentmanner in transgenic tobacco. A second MAR was isolated fromthe genome of Arabidopsis thaliana by preselection for autonomouslyreplicating sequence (ARS) activity in yeast. Flanking copiesof the A. thaliana MAR stimulated median reporter gene expressionin transgenic plants by five to ten fold. Neither MAR significantlyreduced the variation in transgene expression between individualtransformants, or conferred copy number-dependence in gene expression. (Received July 24, 1997; Accepted November 10, 1997)