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Results for “Matrix Attachment Regions”

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PubMed2003

Lentivirus vectors incorporating the immunoglobulin heavy chain enhancer and matrix attachment regions provide position-independent expression in B lymphocytes.

In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (E micro ) with and without associated matrix attachment regions (E micro MAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34(+) progenitors in vitro transduced with E micro - and E micro MAR-containing lentivectors. Lastly, we evaluated the expression from the E micro MAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the E micro and E micro MAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the E micro MAR-containing vector and not other cells types or vectors. Proviral genomes with the E micro MAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the E micro MAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies.

Animals↗

PubMed1994

Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions.

Transcription of the immunoglobulin mu heavy chain locus is regulated by an intronic enhancer that is flanked on both sides by nuclear matrix attachment regions (MARs). These MARs have now been shown to be essential for transcription of a rearranged mu gene in transgenic B lymphocytes, but they were not required in stably transfected tissue culture cells. Normal rates of transcriptional initiation at a variable region promoter and the formation of an extended deoxyribonuclease I (DNase I)--sensitive chromatin domain were dependent on MARs, although DNase I hypersensitivity at the enhancer was detected in the absence of MARs. Thus, transcriptional activation of the mu gene during normal lymphoid development requires a synergistic collaboration between the enhancer and flanking MARs.

Animals↗

PubMed2000

Nuclear matrix attachment regions of human papillomavirus type 16 repress or activate the E6 promoter, depending on the physical state of the viral DNA.

Two nuclear matrix attachment regions (MARs) bracket a 550-bp segment of the long control region (LCR) containing the epithelial cell-specific enhancer and the E6 promoter of human papillomavirus type 16 (HPV-16). One of these MARs is located in the 5' third of the LCR (5'-LCR-MAR); the other lies within the E6 gene (E6-MAR). To study their function, we linked these MARs in various natural or artificial permutations to a chimeric gene consisting of the HPV-16 enhancer-promoter segment and a reporter gene. In transient transfections of HeLa cells, the presence of either of these two MARs strongly represses reporter gene expression. In contrast to this, but similar to the published behavior of cellular MARs, reporter gene expression is stimulated strongly by the E6-MAR and moderately by the 5'-LCR-MAR in stable transfectants of HeLa or C33A cells. To search for binding sites of soluble nuclear proteins which may be responsible for repression during transient transfections, we performed electrophoretic mobility shift assays (EMSAs) of overlapping oligonucleotides that represented all sequences of these two MARs. Both MARs contain multiple sites for two strongly binding proteins and weak binding sites for additional factors. The strongest complex, with at least five binding sites in each MAR, is generated by the CCAAT displacement factor (CDP)/Cut, as judged by biochemical purification, by EMSAs with competing oligonucleotides and with anti-CDP/Cut oligonucleotides, and by mutations. CDP/Cut, a repressor that is down-regulated during differentiation, apparently represses HPV-16 transcription in undifferentiated epithelials cells and in HeLa cells, which are rich in CDP/Cut. In analogy to poorly understood mechanisms acting on cellular MARs, activation after physical linkage to chromosomal DNA may result from competition between the nuclear matrix and CDP/Cut. Our observations show that cis-responsive elements that regulate the HPV-16 E6 promoter are tightly clustered over at least 1.3 kb and occur throughout the E6 gene. HPV-16 MARs are context dependent transcriptional enhancers, and activated expression of HPV-16 oncogenes dependent on chromosomal integration may positively select tumorigenic cells during the multistep etiology of cervical cancer.

Base Sequence↗

PubMed1996

Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state.

The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. We show that the polymerized alpha-helical rod domain of lamin Dm0 provides by itself the specific binding to the ftz M/SAR. In contrast, unpolymerized rod domain does not bind specifically to this M/SAR. Non-specific binding to DNA is also observed with Dm0 containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggest that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state.

Amino Acid Sequence↗

PubMed2003

Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3' MAR.

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.

3' Untranslated Regions↗

PubMed2007

The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants.

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a beta-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chi-MARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chi-MARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.

Animals↗

PubMed1996

Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency.

The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression.

Animals↗

PubMed1998

Flank matrix attachment regions (MARs) from chicken, bean, yeast or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants.

The effect of flanking matrix attachment regions (MARs) on homology-dependent trans-silencing was tested using two strong trans-silencing loci. The transgenic tobacco line 271 carries at a single locus a p35S-RiN-tNos transgene which is able to silence, in trans and at the transcriptional level, the expression of any p35S-driven transgene irrespective of its position. The transgenic tobacco line 6b8 carries at a single locus a p35S-uidA-tRbcS transgene which is able to silence in trans, at the post-transcriptional level, the expression of any uidA-expressing transgene irrespective of its position. Various transgenic tobacco lines carrying a target p35S-uidA-tNos transgene, flanked on each side by MARs from chicken, bean, yeast or tobacco, were crossed with lines carrying the 271 and 6b8 loci. Expression of the target transgene was silenced in all hybrids, irrespective of the presence or absence of MAR sequences. These results therefore demonstrate that MARs are not able to protect transgene expression from strong silencing loci that act in trans.

Animals↗

PubMed1996

Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19.

Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well.

Base Sequence↗

PubMed2005

Matrix attachment regions as targets for retroviral integration.

BACKGROUND: The randomness of retroviral integration has been debated for many years. Recent evidence indicates that integration site selection is not random, and that it is influenced by both viral and cellular factors. To study the role of DNA structure in site selection, retroviral integration near matrix attachment regions (MARs) was analyzed for three different groups of retroviruses. The objective was to assess whether integration near MARs may be a factor for integration site selection. RESULTS: Results indicated that MLV, SL3-3 MuLV, HIV-1 and HTLV-1 integrate preferentially near MARs, specifically within 2-kilobases (kb). In addition, a preferential position and orientation relative to the adjacent MAR was observed for each virus. Further analysis of SL3-3 MuLV insertions in common integration sites (CISs) demonstrated a higher frequency of integration near MARs and an orientation preference that was not observed for integrations outside CISs. CONCLUSION: These findings contribute to a growing body of evidence indicating that retroviral integration is not random, that MARs influence integration site selection for some retroviruses, and that integration near MARs may have a role in the insertional activation of oncogenes by gammaretroviruses.

Animals↗

PubMed2002

Comparison of orthologous and paralogous DNA flanking the wheat high molecular weight glutenin genes: sequence conservation and divergence, transposon distribution, and matrix-attachment regions.

Extended flanking DNA sequences were characterized for five members of the wheat high molecular weight (HMW) glutenin gene family to understand more of the structure, control, and evolution of these genes. Analysis revealed more sequence conservation among orthologous regions than between paralogous regions, with differences mainly owing to transposition events involving putative retrotransposons and several miniature inverted transposable elements (MITEs). Both gyspy-like long terminal repeat (LTR) and non-LTR retrotransposon sequences are represented in the flanking DNAs. One of the MITEs is a novel class, but another MITE is related to the maize Stowaway family and is widely represented in Triticeae express sequence tags (ESTs). Flanking DNA of the longest sequence, a 20 425-bp fragment including and surrounding the HMW-glutenin Bx7 gene, showed additional cereal gene-like sequences both immediately 5' and 3' to the HMW-glutenin coding region. The transcriptional activities of sequences related to these flanking putative genes and the retrotransposon-related regions were indicated by matches to wheat and other Triticeae ESTs. Predictive analysis of matrix-attachment regions (MARs) of the HMW glutenin and several alpha-, gamma-, and omega-gliadin flanking DNAs indicate potential MARs immediately flanking each of the genes. Matrix binding activity in the predicted regions was confirmed for two of the HMW-glutenin genes.

3' Flanking Region↗

PubMed1988

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.

Animals↗

PubMed2004

DNA replication initiates at domains overlapping with nuclear matrix attachment regions in the xenopus and mouse c-myc promoter.

Only a very few origins have been mapped in different multicellular organisms, and they do not share detectable consensus sequence elements. Moreover, it is not clear if origins are localized at similar positions in the corresponding locus in genomes of different organisms. Here, we have mapped DNA replication origins in the c-myc locus both in Xenopus and mouse, allowing a comparison of the corresponding sites in three different animal species (Xenopus, mouse, human). An origin of DNA replication is present in the three homologous c-myc loci. In Xenopus, a main DNA replication origin was located 3 kilobases (kb) upstream of the active c-myc promoter, whereas, in mouse, we detected an origin 1 kb upstream of the promoter, as previously mapped in human c-myc. We also identified a nuclear matrix attachment region in both Xenopus and mouse, which is localized to two different regions of the c-myc promoter region. However, in both cases, the nuclear matrix attachment sites are close to the DNA replication origin mapped in the locus. These data suggest that global features of chromatin organization in different organisms may contribute to DNA replication origin localization.

Animals↗

PubMed1999

Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer-promoter interactions.

The immunoglobulin intragenic mu enhancer region acts as a locus control region that mediates transcriptional activation over large distances in germ line transformation assays. In transgenic mice, but not in transfected tissue culture cells, the activation of a variable region (V(H)) promoter by the mu enhancer is dependent on flanking nuclear matrix attachment regions (MARs). Here, we examine the effects of DNA methylation, which occurs in early mouse development, on the function of the mu enhancer and the MARs. We find that methylation of rearranged mu genes in vitro, before transfection, represses the ability of the mu enhancer to activate the V(H) promoter over the distance of 1.2 kb. However, methylation does not affect enhancer-mediated promoter activation over a distance of 150 bp. In methylated DNA templates, the mu enhancer alone induces only local chromatin remodeling, whereas in combination with MARs, the mu enhancer generates an extended domain of histone acetylation. These observations provide evidence that DNA methylation impairs the distance independence of enhancer function and thereby imposes a requirement for additional regulatory elements, such as MARs, which facilitate long-range chromatin remodeling.

Acetylation↗

PubMed1998

Matrix attachment regions and structural colinearity in the genomes of two grass species.

In order to gain insights into the relationship between spatial organization of the genome and genome function we have initiated studies of the co-linear Sh2/A1- homologous regions of rice (30 kb) and sorghum (50 kb). We have identified the locations of matrix attachment regions (MARs) in these homologous chromosome segments, which could serve as anchors for individual structural units or loops. Despite the fact that the nucleotide sequences serving as MARs were not detectably conserved, the general organizational patterns of MARs relative to the neighboring genes were preserved. All identified genes were placed in individual loops that were of comparable size for homologous genes. Hence, gene composition, gene orientation, gene order and the placement of genes into structural units has been evolutionarily conserved in this region. Our analysis demonstrated that the occurrence of various 'MAR motifs' is not indicative of MAR location. However, most of the MARs discovered in the two genomic regions were found to co-localize with miniature inverted repeat transposable elements (MITEs), suggesting that MITEs preferentially insert near MARs and/or that they can serve as MARs.

Base Composition↗

PubMed2003

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation.

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

Animals↗

PubMed2006

Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of approximately 510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.

Animals↗

PubMed1999

Role of the intronic elements in the endogenous immunoglobulin heavy chain locus. Either the matrix attachment regions or the core enhancer is sufficient to maintain expression.

High level expression in mice of transgenes derived from the immunoglobulin heavy chain (IgH) locus requires both the core enhancer (Emu) and the matrix attachment regions (MARs) that flank Emu. The need for both elements implies that they each perform a different function in transcription. While it is generally assumed that expression of the endogenous IgH locus has similar requirements, it has been difficult to assess the role of these elements in expression of the endogenous heavy chain gene, because B cell development and IgH expression are strongly interdependent and also because the locus contains other redundant activating elements. We have previously described a gene-targeting approach in hybridoma cells that overcomes the redundancy problem to yield a stable cell line in which expression of the IgH locus depends strongly on elements in the MAR-Emu-MAR segment. Using this system, we have found that expression of the endogenous mu gene persists at substantially (approximately 50%) normal levels in recombinants which retain either the MARs or Emu. That is, despite the dissimilar biochemical activities of these two elements, either one is sufficient to maintain high level expression of the endogenous locus. These findings suggest new models for how the enhancer and MARs might collaborate in the initiation or maintenance of transcription.

Animals↗