Journal of Molecular Biology
Volume 274, Issue 2, 28 November 1997, Pages 181-196
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Regular article
Stress-induced duplex DNA destabilization in scaffold/matrix attachment regions 1

https://doi.org/10.1006/jmbi.1997.1385Get rights and content

Abstract

S/MARs are DNA elements 300 to several thousand base-pairs long, which are operationally defined by their affinity for the nuclear scaffold or matrix. S/MARs occur exclusively in eukaryotic genomes, where they mediate several functions. Because S/MARs do not have a clearcut consensus sequence, the characteristics that define their activity are thought to be structural. Ubiquitous S/MAR binding proteins have been identified, but to date no unique binding sequence or structural motif has been found. Here we show by computational analysis that S/MARs conform to a specific design whose essential attribute is the presence of stress-induced base-unpairing regions (BURs). Stress-induced destabilization (SIDD) profiles are calculated using a previously developed statistical mechanical procedure in which the superhelical deformation is partitioned between strand separation, twisting within denatured regions, and residual superhelicity. The results of these calculations show that BURs exhibit a succession of evenly spaced destabilized sites that would render part or all of the S/MAR sequence single stranded at sufficient superhelicity. These analyses are performed for a range of sequenced S/MAR elements from the borders of eukaryotic gene domains, from centromeres, and from positions where S/MARs are known to support the action of an enhancer. The results reported here are in excellent agreement with earlier in vitro chemical reactivity studies. This approach demonstrates the potential for computational analysis to predict the points of division of the eukaryotic genome into functional units (domains), and also to locate certain cis-regulatory sequences.

Introduction

Vertebrate chromatin is organized into loops by periodic attachment to the nuclear scaffold or matrix at positions whose average separations are approximately 60 kb in somatic cells Gasser and Laemmli 1987, Mirkovitch et al 1987, Vogelstein et al 1980, and 27 kb in the male haploid genome (Barone et al., 1994). The DNA elements mediating this attachment have been termed scaffold-attached or matrix associated regions (S/MARs). S/MARs are unique features of eukaryotic genomes, as demonstrated by the observation that S/MAR-scaffold interactions cannot be disrupted by an up to 60,000-fold excess of double stranded bacterial DNA (Kay & Bode, 1995). Recently it has been demonstrated that centromeric regions have clusters of attachment sites concentrated along the chromosomal axis (Strissel et al., 1996).

S/MAR elements are associated with a variety of biological functions, apparently in consequence either of their structure and/or of their interactions with proteins. Experiments suggest they play important roles during gene expression. S/MARs are thought to support cell type-specific expression of genes Bonifer et al 1994, McKnight et al 1992, McKnight et al 1996 and have been implicated in gene switching during development (Boulikas, 1995). Evidence from several laboratories shows that some S/MARs coexist with enhancers (Gasser & Laemmli, 1986). This association is particularly intriguing, as most or all S/MARs also have the capacity to augment transcription via a non-enhancer mechanism (for a review, see Bode et al., 1995). The most thoroughly studied example involves the immunoglobulin κ and μ-chain intronic enhancers, which are associated with one and with two distinct S/MAR elements, respectively Cockerill and Garrard 1986a, Cockerill and Garrard 1986b, Cockerill et al 1987. They function in domain opening Bode et al 1996, Kas et al 1993, which occurs in forming accessible chromatin domains during embryonic development Forrester et al 1994, Jenuwein et al 1993. The regional demethylation that must occur as a prerequisite for opening utilizes distinct cis-acting modules, including the intronic enhancer element and the S/MAR Jenuwein et al 1997, Lichtenstein et al 1994. While any S/MAR sequence appears to be able to function in this reaction, tissue specificity is mediated by sequences within the intronic enhancer (Kirillov et al., 1996). Some, and possibly all, of these enhancer-related activities are regulated by specific S/MAR binding factors. Among these, the nuclear factor-μ negative regulator (NF-μNR) and the B cell regulator of IgH transcription (BRIGHT) show a reciprocal lymphoid expression pattern. NF-μNR is expressed in non-B cells, where it attenuates the enhancer Scheuermann and Chen 1989, Zong and Scheuermann 1995, while BRIGHT upregulates IgH expression in mature B cells (Herrscher et al., 1995). The sites specific for BRIGHT also are recognized by SATB1, which was the first protein for which a S/MAR-related association was noted Dickinson et al 1992, Herrscher et al 1995.

Possible roles for S/MARs in replication are suggested by several observations. ORI sites mapped in various eukaryotic genomes appear to coincide with S/MARs. In yeast, chromosomal replication initiates at a subset of the ARS elements present in the genome for which S/MAR functions have been demonstrated (Amati et al., 1990). In Drosophila there also is a close correlation between ARS and S/MAR activity (Amati and Gasser, 1990). Although in most assays in higher eukaryotes replication initiation seems to occur in broad regions, an overlap between ORI and S/MAR functions at the DHFR replication origin has been demonstrated (Dijkwel and Hamlin, 1995). Active chromosomal ORIs are permanently associated with the matrix (Carri et al., 1986). DNA is reeled through the replication machinery, which is a part of the matrix (Hozak & Cook, 1994).

To date, the identification of genomic regions associating with the nuclear matrix has relied primarily on biochemical studies. DNA segments with an affinity for the nuclear matrix have been recovered by a variety of assays (for reviews, see Boulikas 1995, Kay and Bode 1995). The associations of active genomic regions with the scaffold are mediated both by S/MARs and by a distinct preference of the nuclear matrix for supercoiled DNA as it occurs during transcription Kay and Bode 1994, Tsutsui et al 1988. Assays designed to monitor attachment differ in their ability to recover constitutive and transcription-dependent forms of DNA association.

Sequence searches have been inadequate at revealing putative scaffold attachment sites because, although several characteristic motifs are known, including A+T-richness, no unique consensus S/MAR sequence has been found Boulikas 1993, Kramer and Krawetz 1995, Kramer et al 1996. A map of the S/MARs on human chromosome 19 supports the view that these sites do not share any common repeat sequences (Nikolaev et al., 1996).

S/MAR functional activity may be related instead to topological or structural features that are not strictly linked to primary sequence. Structural properties which commonly occur in A+T-rich regions include natural curvature, a narrow minor groove in oligo(dA) tracts, and a susceptibility to denature Bode et al 1995, Boulikas 1993, Boulikas 1995. Chemical probes and two dimensional gel analyses of S/MAR regions placed in plasmids under superhelical tension show that these elements readily relieve imposed torsional strain by stable base-unpairing. In all the cases that have been experimentally analyzed to date, this unpairing initiated at a nucleation site, here called the core unwinding element or CUE Bode et al 1992, Bode et al 1995, Bode et al 1996. Although all S/MAR sequences appear to share a propensity to form non-B DNA structures under superhelical tension (Boulikas, 1993), in some cases their sequence suggests that this could be either a triple helical H-form or a denatured region. In both structures at least one unpaired strand is present.

S/MAR activities are thought to be mediated by distinct sets of DNA binding proteins that recognize specific structural features such as single strands (Bode et al., 1996). Support for this hypothesis comes from observations that decreases in the thermodynamic stability of S/MAR regions correlate both with increases in their strength of binding to nuclear scaffold/matrix preparations in vitro, and with their potential to augment transcriptional initiation rates in vivo Allen et al 1996, Bode et al 1992, Mielke et al 1990, Schubeler et al 1996.

Here, we perform a statistical mechanical analysis of S/MARs within genomic sequences to predict their stress-induced duplex-destabilization (SIDD) properties. This procedure has proven to be highly accurate in finding sites of denaturation and predicting the extent of opening at each site, as detected by the Kowalski nuclease assay Benham 1992, Benham 1993, Benham 1996a, Kowalski et al 1988. Several specific sequenced S/MARs are analyzed, for which in vitro data on base-unpairing and matrix binding are available Bode et al 1992, Kohwi-Shigematsu and Kohwi 1990, Mielke et al 1990. This analysis finds that those locations having S/MAR activity also exhibit strong destabilization patterns with characteristic lengths and structures. The accuracy of these results is assessed by comparison with experimental data on base-unpairing within, and functional activity of, these S/MAR regions.

Section snippets

Calculations

At thermodynamic equilibrium a population of identical superhelical DNA molecules will be distributed among its available conformational states according to Boltzmann’s law. If the states are indexed by i, and if the free energy of state i is Gi, then the governing partition function is given by:Z=i e(−Gi/RT) where R is the gas constant and T is the absolute temperature. The equilibrium probability pi of state i, which is its fractional occupancy in a population at equilibrium, equals the

The immunoglobulin heavy chain enhancer-associated S/MARs: a paradigm?

Kohwi-Shigematsu & Kohwi (1990) have demonstrated an overlap between binding sites for the regulator NF-μNR and locations that become stably and uniformly unpaired when the IgH gene region is subjected to torsional stress. Prominent destabilized sites coincide with the strong 3′- S/MAR and the weaker 5′-S/MAR of the IgH enhancer (Cockerill et al., 1987; cf. elements XVIl and XVIr in Mielke et al., 1990).

We have evaluated the destabilization properties of this region when it is inserted in the

Base-unpairing in vivo and in vitro

Previous work has shown that base-unpairing regions (BURs) are closely associated with several types of DNA functional elements. BURs characterized by their reaction properties with single strand-specific enzymes or reagents have been reported to occur in vivo within the transcriptional regulatory regions of several genes, including the chicken βA-globin gene (Kohwi-Shigematsu et al., 1983), the human CMV major immediate early gene (Kohwi-Shigematsu & Nelson, 1988), the interferon-β gene (Bode

Acknowledgements

This work was supported in part by grants to C. J. B. from the National Institutes of Health and from the National Science Foundation, and to J. B. O. from the Deutsche Forschungsgemeinschaft Bo419/6-1.

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