The tethering of DNA to the nuclear matrix plays a vital role in transcription 92122. Using T-cell differentiation as a model we will describe how MARs facilitate transcription and reveal how they shape chromatin architecture to insulate chromatin domains from the effects of flanking chromatin.

Upon stimulation by antigen, naive CD4 helper T cells differentiate into effector Th1 and Th2 cells. In mice, Ifng (the gene for the cytokine interferon-γ) is silenced in naive T cells but transcribed in activated Th1 cells. The architecture of the Ifng locus has been analyzed in these two cell types by a combination of chromosome conformation capture and microarray technology 22. In naive T cells Ifng was found to exist in a linear conformation, but in Th1 cells it is present in a chromatin loop, due to tethering of DNA to the nuclear matrix by MARs 7 kb upstream and 14 kb downstream of the locus. The absence of this selective DNA attachment to the nuclear matrix in naive T cells suggests that dynamic DNA anchors mediate the formation of the looped structure and the expression of the Ifng locus 22.

The molecular mechanisms by which MARs reorganize higher-order chromatin structure have been investigated in detail at the murine Th2 cytokine locus, which contains the cluster of coordinately regulated genes Il4, Il13 and Il5 in a region of about 120 kb 23. These genes are expressed in Th2 cells but are silent in naive T cells. Following Th2 activation, expression of the nuclear matrix protein SATB1 is rapidly induced, and MARs within the locus mediate the formation of small loops by anchoring the loops onto a common protein core associated with SATB1 12. Down-regulation of SATB1 expression by RNA interference prevents both the formation of this looped structure and transcriptional activation of the locus 12. In SATB1-null thymocytes (developing T cells) the expression of many genes is spatially and temporally misregulated, and T-cell development in SATB1-deficient mice is prematurely blocked. These results indicate that the binding of SATB1 at MARs regulates the expression of T-cell differentiation genes by reorganizing higher-order chromatin architecture 2425. A similar MAR-mediated loop-formation mechanism regulates expression of the human β-globin gene cluster 2627.

Cai et al. 25 reported that SATB1 recruits several chromatin-remodeling enzymes at MARs to activate or repress the expression of nearby sequences. Other studies have shown that MARs interact dynamically with basal components of the transcription machinery and with splicing factors 2829. In eukaryotic cells, mRNA synthesis is concentrated at discrete transcription 'factories' or foci within the nucleus, which contain RNA polymerases, RNA transcripts, transcription factors and mRNA-processing factors 30. The retention of RNA polymerase II and general transcription factors in nuclei after extraction of soluble proteins and nuclease digestion suggests that transcription factories are assembled onto the nuclear matrix 3132. As MARs associate with components of transcription factories as well as the nuclear matrix, it is tempting to speculate that dynamic interactions between MARs and the matrix bring together proximal and distal regulatory sequences and localize them close to transcription factories, thus promoting efficient regulation of gene expression (Figure 1).

Many genes are known to be shielded by so-called 'insulator' elements from stimulatory or repressive effects attributable to the chromatin state and regulatory elements in flanking regions. MARs commonly map to sequences flanking genes, and co-localize with some of the most extensively analyzed insulator elements, including gypsy, a retrotransposon in Drosophila melanogaster, suggesting that MARs have an insulator function 33. In Drosophila, the nuclear matrix protein Su(Hw) binds to gypsy, creating chromatin loops 34. Certain mutations in Su(Hw) that disrupt the loop structures render the insulator non-functional 3435. This suggests that the tethering of MARs to the nuclear matrix topologically constrains the DNA into looped structures, protecting the intervening DNA from the influence of cis-regulatory elements outside the loop. In vertebrates, CTCF, a ubiquitous nuclear matrix protein, binds to insulators and has also been shown to interact with MARs 36. While the precise mechanisms of CTCF insulation remain unclear, the binding of CTCF to MARs might block interactions between promoters and unrelated enhancers and create looped structures that delimit different chromosomal domains 37. Experiments in a wide variety of higher eukaryotes have shown that in stably transfected cells, MAR-containing transgenes were expressed at higher levels compared with transgenes lacking MARs, indicating that the MARs shield the transgenes from the effects of the neighboring host chromatin 3839.

Taken together, the experimental evidence described above supports the view that MARs function as landing platforms for a wide range of matrix proteins. Such interactions form complex higher-order nucleoprotein structures, which insulate chromatin domains and also control gene expression by forming bridges between components of the basal transcription machinery and distal and proximal regulatory elements. MARs can thus be defined as cis-acting elements constituting a critical layer of transcriptional regulation.

To ensure that the genome is copied accurately, and only once per cell cycle, eukaryotes have evolved intricate mechanisms to regulate DNA replication. Some of the best-characterized origins of replication (ORIs) have been mapped to AT-rich genomic regions with base-unpairing elements. Futhermore, sequences at or near the ORIs for the human lamin B2 gene, the Chinese hamster dihydrofolate reductase β and β' genes, the human β-globin gene, the chicken α-globin and lysozyme genes, and the Xenopus and mouse c-myc genes, function as dynamic MARs during the cell cycle 40414243444546.

These findings are in agreement with observations that DNA replication is temporally and spatially ordered in the nuclei of animal cells. Several replicons are coordinately replicated at foci in the S-phase nucleus 4748. Evidence that replication foci are associated with the nuclear matrix came first from electron microscopy 49. Further support came from a study of nuclear matrix structures where DNA synthesis occurred at replication sites that were indistinguishable from those found in intact cells 50. Radichev and colleagues 51 found that DNA replication initiates at discrete chromosomal sites attached to the nuclear matrix.

At replication foci, the nuclear matrix houses factors necessary for DNA replication, such as DNA polymerases, the sliding clamp (PCNA) and single-strand binding protein (RPA), and provides structural support throughout the replication process. Wu and Gilbert 52 proposed that origins are selected and replicon size is determined in early G1 phase of the cell cycle. Using an in vitro system, it was subsequently shown that MCM2, a component of the pre-replicative complex, is loaded onto chromatin gradually and cumulatively throughout G1, but is rapidly excluded from active replication foci in S phase 53. Tatsumi and colleagues 54 reported a similar cycle of events for ORC1, a component of the replication initiation complex at ORIs. This coincides with recruitment of the chromatin-bound ORC2-5 complex to a structure likely to be the nuclear matrix 55, suggesting a link between the accumulation of ORC1 and the assembly of the replication complex in human nuclei.

These observations fit a model in which MARs stably anchor the replicon ends and, during G1, small-scale sub-chromosomal chromatin refolding recruits ORIs to the nuclear matrix, where factors accumulate to form the pre-replicative complexes (Figure 2). Subsequently, as ORIs begin to replicate in S phase, certain protein factors dissociate from the chromatin and undergo proteolysis - as part of a control mechanism to prevent re-replication - thus releasing the ORIs from the nuclear matrix. In the meantime, replication continues at the initial location as DNA is reeled through the replication machinery or replication factory 49. At the ends of replicons, stable MARs could act as barriers between adjacent replicons by preventing the accumulation of supercoiled DNA structures, while providing binding sites for topoisomerase II, which can resolve replication intermediates.

Integration of retroviral DNA into the host genome is essential for viral replication. Although retroviral integration sites lack a consensus sequence, they are often AT-rich with base unpairing and DNA-bending and unwinding elements 5657. DNA sequence analysis indicates that both DNA tumor viruses and retroviruses integrate within or close to MARs (Figure 3) 5859. Furthermore, the efficiency of transcription of the retrovirus HIV-1 is determined by the proximity of its integration to MARs 57. As SATB1 binds to MARs flanking HIV-1 integration sites and silencing of SATB1 gene expression alters the pattern of integration sites, it has been suggested that retroviruses use MARs to form viral pre-integration complexes 60.

MARs also appear to play a role in some cancers. Chromosome rearrangements are hallmarks of certain malignancies and inherited genetic disorders. The breakpoints of recurrent translocations in leukemia as well as deletions involving the breast-cancer susceptibility genes BRCA1 and BRCA2 occur at MARs, indicating that the bringing together of these sequences at the nuclear matrix facilitates their illegitimate recombination 6162. Patients who develop leukemia following treatment of a primary tumor with inhibitors of topoisomerase II often have specific chromosome translocations in their cancer cells whose breakpoints contain MARs, emphasizing the importance of the chromatin environment in the generation of chromosome aberrations 6364.

Fragile sites are hypervariable regions that generate genomic instability in tumors. Certain fragile sites contain long AT-rich minisatellites, called AT-islands, which function as MARs 65. AT-islands are susceptible to considerable repeat expansion, which, in the fragile site FRA16B associated with leukemia, appears to strengthen their attachment to the nuclear matrix 65. The presence of abnormal transcripts of the tumor suppressor gene WWOX (which spans FRA16B) in the absence of detectable mutations or deletions may be caused by aberrant chromatin architecture due to enhanced MAR anchoring by expanded AT-islands 66.

Identification of AT-islands has led to the emergence of a new class of drugs that specifically alkylate them 67. These drugs exhibit an extraordinary cytotoxicity, which is likely to be due to their disruption of replication and transcription, the two essential nuclear processes organized at MARs (Figure 4). One of these drugs, bizelesin, binds specifically to the minor groove of DNA at AT-rich regions and generates interstrand crosslinks. It has high cytotoxic activity in vitro towards a broad spectrum of human cancer cell lines and, more importantly, high activity against various tumors engrafted in mice 6869. While extensive development will be needed to make these compounds safe anti-cancer drugs for clinical use, their DNA-sequence specificity might offer a novel approach for targeting tumor cells containing expanded AT-repeat sequences.

Our understanding of how the genome functions in the context of the nucleus has been propelled by indisputable evidence that distinct genomic sites bind to regulatory proteins at the nuclear matrix. The emerging picture is that these genomic anchors regulate transcription and replication by dynamically organizing chromatin in three-dimensional space. The recognition that these essential nuclear processes are compartmentalized into microenvironments that are compromised in diseases such as cancer 70 emphasizes the need to define chromatin architecture more accurately in relation to the various nuclear domains. In reaching beyond the linear genome, we will approach a more comprehensive view of genomic function and are likely to identify truly novel targets for therapy.