(Received for publication, October 20, 1994; and in revised form, July 13, 1995)
From the
The immunoglobulin heavy chain (IgH) intronic enhancer stimulates transcription from functional promoters in B lymphocytes but not other cell types. The observation that binding sites for the nuclear factor-µ negative regulator (NF-µNR) enhancer repressor overlap nuclear matrix attachment regions (MARs) in this enhancer has lead to the hypothesis that the cell type specificity of the enhancer might be controlled by regulating nuclear matrix attachment (Scheuermann, R. H., and Chen, U.(1989) Genes & Dev. 3, 1255-1266). To understand the role of MARs in IgH enhancer regulation, we have identified a novel MAR-binding protein, MAR-BP1, from soluble nuclear matrix preparations based on its ability to bind to the MARs associated with the IgH enhancer. Purified MAR-BP1 migrates as a 33-kDa protein, and it can be found in nuclear matrix preparations from a number of different types of lymphoid cell lines. Although specific binding sites have been difficult to localize by chemical or enzymatic footprinting procedures, NF-µNR binding sites are critical for efficient MAR-BP1 binding. Indeed, binding of the IgH enhancer to either intact nuclear matrix preparations or to MAR-BP1 is mutually exclusive to NF-µNR binding. These results are consistent with a model for cell-type specific regulation in which binding of the NF-µNR repressor to the IgH enhancer prevents nuclear matrix attachment in inappropriate cells by interfering with MAR-BP1/enhancer interaction.
Transcription of the immunoglobulin heavy chain (IgH) ()gene is influenced by at least two distinct classes of
cis-acting elements, promoters and enhancers. The IgH intronic
enhancer, located between the J
elements and Cµ,
stimulates transcription from promoters in a relatively distance- and
orientation-independent manner, and acts as a cell type-specific
regulator, being functional in B cells and nonfunctional in non-B cells
(for review, see Staudt and Lenardo(1991)). This enhancer contains
multiple DNA sequence motifs that function as interaction sites for
sequence-specific DNA-binding proteins. Many of these sites have been
demonstrated to be important for transcriptional regulation. The B cell
specificity of the IgH enhancer is regulated by both positively and
negatively acting B cell-specific enhancer elements as well as
protein-protein interactions between B cell-specific and ubiquitously
expressed nuclear factors.
We have identified negative elements in the IgH enhancer that are bound by a nuclear protein, nuclear factor-µ negative regulator (NF-µNR). NF-µNR purified from a preB cell line binds to four sites flanking the enhancer core in a cooperative manner (Scheuermann, 1992). NF-µNR activity is expressed in non-B cells and appears to repress the enhancer in these inappropriate cells (Scheuermann and Chen, 1989). One intriguing aspect of NF-µNR binding is that the four binding sites in the IgH enhancer overlap two nuclear matrix attachment regions (MARs) (Cockerill et al., 1987).
MARs are AT-rich DNA sequences that remain attached to a salt-resistant nuclear structure referred to as the nuclear matrix or scaffold (Berezney and Coffey, 1974). MARs/scaffold attachment regions have been mapped to DNA segments that are several hundred base pairs in length (Gasser and Laemmli, 1986; Cockerill and Garrard, 1986), which anchor chromatin loops to the nuclear matrix (for review, see Laemmli et al., 1992). Juxtaposition or cohabitation of MARs with transcriptional regulatory elements suggests that MARs may influence transcription (Cockerill et al., 1987). Indeed, some MAR sequences have been shown to stimulate transcription in transfection studies (Stief et al., 1989; Xu et al., 1989; Phi-Van et al., 1990; Klehr et al., 1991; Bode et al., 1992). Furthermore, the importance of the IgH enhancer MARs to enhancer activity has recently been demonstrated in transgenic mouse experiments where constructs lacking these MARs were inactive (Forrester et al., 1994).
Several MAR-binding proteins have been identified and purified from nuclear extracts (von Kries et al., 1991, 1994; Tsutsui et al., 1993; von Kries et al., 1994). Some of these proteins require relatively long DNA fragments for efficient binding and may recognize certain structural features of DNA rather than a precise nucleotide sequence (Zhao et al., 1993). Others may have the properties similar to transcription factors (Dickinson et al., 1992; Bidwell et al., 1993), since they recognize specific sequences within DNA fragments. Proteins binding to MARs include lamin B1 and topoisomerase II, which are major components of the nuclear matrix (Luderus et al., 1992; Sperry et al., 1989); the yeast RAP-1 factor and HeLa SAF-A/hnRNP-U, which induce DNA loop formation (Hofmann et al., 1989; Romig et al., 1992; Fackelmayer et al., 1994); and SATB1 and nucleolin, which seems to preferentially bind DNA with base-unpairing potential (Dickinson and Kohwi-Shigematsu, 1995).
Here we describe the identification and purification of a novel 33-kDa nuclear matrix protein, MAR-BP1, which binds to the IgH enhancer MARs. Binding of MAR-BP1 to the IgH enhancer MARs requires the four NF-µNR binding sites. Indeed, binding of MAR-BP1 and NF-µNR to IgH enhancer fragments is mutually exclusive. These results are consistent with a model for cell type-specific regulation in which NF-µNR binding to the IgH enhancer prevents nuclear matrix attachment by interfering with MAR-BP1/enhancer interaction.
In protein competition experiments, P-labeled DNA fragments (
2 ng) were preincubated with
purified NF-µNR, purified MAR-BP1, or soluble matrix proteins and 5
µg of sonicated E. coli DNA as a nonspecific
competitor for 30 min at room temperature. After the additions of 15
µg sonicated E. coli DNA and 5 µg nuclear matrix, the
solution was incubated shaking for one hour at 23 °C and processed
as described above.
Figure 1:
Specific binding of IgH
enhancer to the nuclear matrix. A, MAR sequences and
NF-µNR binding sites in the IgH enhancer fragment. The entire
tissue-specific IgH enhancer is contained within a 1.0-kb XbaI
restriction fragment. The fragment can be subdivided by digestion with PvuII and EcoRI giving the 5`-En, enhancer core, and
3`-En fragments. The MARs located 5` and 3` of the enhancer are shown
by hatchedbars. The four binding sites of the
NF-µNR enhancer repressor protein flanking the enhancer core are
indicated by the openrectangles (P1-P4). Other sites thought to be
important for enhancer function (see Staudt and Lenardo(1991)) are also
indicated, including E boxes (1-5), binding sites for
the nuclear protein Ig-EBP (E and E`), the octamer (O) and µB sites, and consensus sequences for nuclear
matrix attachment (M). B, nuclear matrix attachment
of enhancer fragments. Mixtures of P-labeled DNAs,
including pUC19, a fragment derived from the Ig µ heavy chain
constant region (µ1.2), the 0.92-kb
light chain MAR (
MAR), and the 1.0-kb XbaI IgH enhancer (IgHE) fragment, were incubated with insoluble nuclear matrix
preparations in the presence or absence of E. coli competitor DNA as indicated. The
matrix-associated DNA using mixtures of the IgH enhancer and pUC19 (lanes1-3), the
MAR, and pUC19 (lanes4-6) and the IgH enhancer and µ1.2 (lanes7-9) was analyzed by agarose gel
electrophoresis. In each case, binding is compared with lanes
containing 25% of the DNA input (IP) used in each reaction (lanes1, 4, and 7).
Nuclear matrix
preparations from a B cell line were found to specifically bind IgH
enhancer DNA in the presence of an excess of nonspecific E. coli competitor DNA (Fig. 1B; compare lane2 with 3 and lane8 with 9). In the same samples, the competitor DNA completely
abrogated matrix binding to two control DNA fragments that lack MARs,
the plasmid pUC19 (lane3) and a fragment derived
from the IgH Cµ region (lane9). The MAR
derived from the Ig light chain
gene J-C intron also binds to the
nuclear matrix, apparently with a higher binding affinity (lane6). MARs derived from a Drosophila histone gene
(Dr MAR) and from an IgH chain variable region gene promoter (Webb et al., 1991) also bind to these nuclear matrix preparations
with similar binding affinity as the enhancer MARs (data not shown).
Figure 2:
Identification of a soluble MAR-binding
protein. A, 0.5-ng P-labeled 5`-En DNA was
incubated with 0.5 µg of soluble matrix protein, and the
protein-DNA complexes were analyzed by MSA. Lane1 contains the labeled probe without matrix protein; lanes3-9 contain a 200-fold molar excess of unlabeled
DNA competitor as indicated, except that poly(dA/dT) was used in a
2000-fold molar excess. The MAR-BP1
DNA complex is indicated with
an arrow. B, as in A except that the 3`-En
fragment was used as a labeled probe.
Using the 3`-En MAR-containing fragment as a probe, a similar pattern was observed (Fig. 2B). Although several faint complexes were observed in addition to the major MAR-binding complex, they are nonspecific since they were competed by the enhancer core and poly(dA/dT). Our data indicate that the same MAR-binding protein is responsible for the complexes seen with 5`-En and 3`-En probes, and yet the complex formed with the 3`-En fragment migrates more slowly than the 5`-En complex. This may be due to the ability of this MAR-binding protein to bend the bound DNA fragments (data not shown). Bending tends to slow the mobility of protein-DNA complexes most dramatically when the binding site is situated in the center of the fragment (Kim et al., 1989). Perhaps the binding site for this MAR-binding protein is situated in the middle of the 3`-En fragment but close to one of the ends of the 5`-En fragment. Changes in mobility of bent DNA are also more dramatic with smaller DNA fragments.
Figure 3:
Purification of a soluble MAR-binding
protein. A, purification fractions containing MAR-binding
proteins were analyzed by SDS-polyacrylamide gel electrophoresis (10%)
and silver staining. Lane1 contains silver staining
marker (Bio-Rad); lane2 contains 1 µg of the
nuclear matrix; lane3 contains denatured nuclear
matrix pellet; lane4 contains 1 µg of soluble
nuclear matrix protein; lane5 contains 0.1 µg of
the DEAE peak fraction; lane6 contains 10 ng of the
peak fraction from DNA-cellulose affinity chromatography. B,
UV cross-linking of MAR-BP1. Internally labeled 5`-En (lanes1, 3, and 4) or 3`-En (lane2) fragments were used in MSAs, the wet gels were
irradiated, specific complexes were isolated, and proteins were
resolved by SDS-polyacrylamide gel electrophoresis. Samples for lanes1 and 2 contained 0.1 µg of
MAR-BP1 DEAE fraction; lane3 contained no protein; lane4 contained 10 ng of NF-µNR fraction
F. Molecular masses (kDa) of protein size markers are
indicated. Similar results were obtained using total soluble nuclear
extracts and DNA-cellulose column
fractions.
In order to determine if this protein is indeed responsible for MAR binding, UV-cross-linking experiments were performed to determine the molecular mass of the MAR-binding activity present in this purified fraction (Fig. 3B). Lanes1 and 2 show that the same protein binds to both 5`-En and 3`-En fragments. The molecular mass of this labeled species is consistent with a 33-kDa protein bound to a small stretch of DNA predicted to remain following DNase digestion of the cross-linked complex. Cross-linking of the 5`-En fragment with purified NF-µNR gives rise to a larger labeled species (lane4), consistent with its 40-kDa molecular mass (Scheuermann, 1992). These results support the contention that although these binding activities interact with the same enhancer fragments, they are distinct. We have named this MAR-binding protein, MAR-BP1, for matrix attachment region binding protein 1.
The cell type specificity of MAR-BP1 expression was
investigated by evaluating MAR binding activity in soluble nuclear
matrix preparations isolated from preB, mature B, and mature T cell
lines by MSA (Fig. 4). DNAMAR-BP1 protein complexes were
found using soluble matrix preparations from all six cell lines tested.
At this point, the significance of lower MAR-BP1 expression in preB
cell lines as compared with mature B cell lines is not clear, but it is
a consistent finding with both preB cell lines examined.
Figure 4: Cell type distribution of MAR-BP1. Soluble nuclear matrix proteins were prepared from the different cell lines as indicated. MAR-BP1 DNA binding activity was examined by MSA with 0.5 µg of protein and 0.5 ng of 5`-En probe. The arrow indicates the characteristic MAR-BP1 complex.
Since the precise mapping of MAR sites was
problematic, we decided to concentrate on approaches to investigate the
relationships between NF-µNR binding sites and MAR-BP1 binding
using MSAs. The 381-bp 5`-En, containing the NF-µNR binding sites
P1 and P2, can be subdivided into smaller fragments by restriction
enzyme digestion (Fig. 5A). No binding could be
demonstrated with purified MAR-BP1 using fragments derived from either
end of the 5`-En fragment (Fig. 5B, S-P and X-R). Specific binding could be demonstrated
to a subfragment containing the middle region (R-P).
This result suggests that the putative MAR-BP1 binding site lies within
the 185-bp region between the RsaI and the SspI
sites; this region also contains the NF-µNR binding sites P1 and
P2. The fact that the binding affinity for this subfragment was only
20% of that of the whole fragment suggests that either the binding
site had been partially disrupted by restriction enzyme cleavage or
that the length requirement for high affinity binding discussed above
is coming into play. Similar results were obtained in experiments using
3`-En fragments as probes, i.e. only the whole 309-bp fragment
had MAR-BP1 binding activity; any further digestion of this fragment
lead to a dramatic loss of binding activity (data not shown).
Figure 5:
MAR-BP1 binding requires NF-µNR sites. A, 381-bp 5`-En fragment (X-P) was digested
with restriction enzymes and subfragments isolated as indicated. B, MAR-BP1 binding to the probes described in A was
evaluated by MSA, quantified by PhosphorImaging, and plotted as a
percentage of total probe in the reaction mix. C, different
NF-µNR binding site deletion mutants were used in MSA competition
experiments to test the effect of NF-µNR binding sites on MAR-BP1
binding. P-labeled 5`-En was incubated with 5 µl of
MAR-BP1 DEAE fraction in the presence of a 10-100 fold molar
excess of competitor DNA from wild-type or mutant IgH enhancer
containing deletions of NF-µNR binding sites P1, P2, P3, and P4
(mut). D, wild-type, P1, or P2 deletion mutant (d1-5`En and d2-5`En) 5`-En DNAs were
used as competitors at a 50-fold molar excess in binding reactions
containing labeled 5`-En probe (lanes1-6) as
described above. Wild-type, P3, or P4 deletion mutants were used in
competition experiment containing labeled 3`-En probe (lanes7-11). E, binding affinities of MAR-BP1 to
wild-type or mutant 5`-En fragments. Binding of varying concentrations
of MAR-BP1 to DNA probes (25 pM) was measured by MSA. The DNA
probes included the 381-bp 5`-En fragment (opensquares), the 301-bp MboII 5`-En fragment (closedsquares), the 350-bp d1-5`En fragment (closedcircles), and the 330-bp d2-5`En
fragment (opencircles). The dissociation constant (K
) was calculated from the protein
concentration required for a 50% shift of the probe (Koudelka, et
al., 1987; Dickinson and Kohwi-Shigematsu, 1995) to be 1.5
10
M for both the 5`-En and the MboII 5`-En fragments. The affinities for the d1-5`En
and d2-5`En fragments were estimated to be at least 5-fold lower
than that of the wild-type 5`En fragment based on the relative amounts
of protein needed to bind 10% of the respective
probes.
Since MAR-BP1 binds to both the 3`-En fragment and to subfragments of 5`-En containing NF-µNR binding sites, the importance of the four NF-µNR sites P1-P4 was investigated. In MSA experiments using the 5`-En fragment as a probe, IgH enhancer fragments in which all four NF-µNR binding sites had been deleted were considerable less effective at competition than the wild-type enhancer (Fig. 5C). In addition, deletion of either P1 or P2 from the 5`-En fragment, or deletion of either P3 or P4 from the 3`-En fragment significantly reduced their ability to compete with the respective wild-type fragments for MAR-BP1 binding (Fig. 5D). These results indicate that NF-µNR binding sites are essential for MAR-BP1 binding, due to either their identity or extensive overlap.
To further analyze the influence of
NF-µNR sites on MAR-BP1 binding, we measured the DNA binding
affinity using 5`-En fragments with or without either P1 or P2 site
deletion (Fig. 5E). The calculated dissociation (K) constant for binding to the intact 5`-En
fragment is 1.5
10
M. However
deletion of either P1 or P2 site dramatically decreases the MAR-binding
affinity by a factor of 5 or more. This reduction in affinity is not
simply due to a shortening of the DNA probe size, since removal of 80
bp from the 5` end by MboII digestion had no effect on binding
affinity. Taken together these results indicate that NF-µNR binding
sites play a critical role for MAR-BP1 binding.
A comparison of the binding affinities for MAR-BP1 and other DNA-binding proteins (Table 1) indicates that although the affinity of MAR-BP1 for MAR fragments is lower than that measured for several transcriptional regulatory proteins like NF-µNR, LEF-HMG, SP1, and MLTF, it is similar to the affinity measured for another MAR-binding protein, nucleolin.
Figure 6:
NF-µNR inhibits nuclear
matrix/enhancer interaction. A, P-labeled IgH
enhancer and the Cµ control fragments were analyzed for specific
binding to insoluble nuclear matrix preparation (5 µg) as described
in Fig. 1(lanes2-4). For samples in lanes3 and 4, NF-µNR (20 ng) was
preincubated with the DNA for 30 min before the addition of insoluble
nuclear matrix. Lane1 contains input DNA (25%). B, the experiment was performed as in A except that
either 0.2 µg of total soluble nuclear matrix (lane3) or 50 ng of purified MAR-BP1 (lane4) was used in place of
NF-µNR.
The requirement
of NF-µNR sites for MAR-BP1 binding suggests that the interaction
of these two proteins might be mutually exclusive. To address this
possibility, a kinetic experiment was performed to examine NF-µNR
binding to a mixture of free DNA and DNAMAR-BP1 complexes (Fig. 7). With time, the amount of NF-µNR
DNA complex
increases until equilibrium is reached (by
15 min). The increase
in NF-µNR
DNA complex is balanced by a decrease in the amount
of free probe remaining (lanes5 and 6). In
contrast, the amount of MAR-BP1
DNA complex remains unchanged.
This result indicates that MAR-BP1 binding prevents NF-µNR
interaction. However, if excess NF-µNR is used and the reaction is
allowed to proceed for 60 min, well beyond the half-life of the
MAR-BP1
DNA complex (25 min), NF-µNR will replace MAR-BP1 (Fig. 8). This result is consistent with the higher affinity of
NF-µNR for this fragment. Thus, in cells where both NF-µNR and
MAR-BP1 are present (e.g. non-B cells) the enhancer would be
occupied by NF-µNR.
Figure 7: MAR-BP1 prevents binding of NF-µNR to the 5`-En fragment. MAR-BP1 (DEAE fraction, 20 ng) was incubated with the labeled 5`-En fragment for 30 min to reach equilibrium. NF-µNR (2 ng) was then added to each reaction, and samples were loaded onto an MSA gel at the indicated times.
Figure 8: NF-µNR displaces DNA-bound MAR-BP1. MAR-BP1 DEAE fraction (20 ng, lane2; 25 ng, lane3; 30 ng, lane4; 40 ng, lanes5-9) was incubated with the labeled 5`-En fragment for 30 min at room temperature to reach equilibrium, at which time purified NF-µNR was added at 2 ng (lane6), 3 ng (lane7), 3.5 ng (lane8), and 4 ng (lane9). Incubation was continued for an additional 60 min on ice before MSA analysis.
Based on its size, MAR-BP1 is distinct from these members of the MAR-binding family of nuclear proteins. However, two proteins that have been found to bind MARs have similar sizes, NF-µNR and histone H1. A variety of observations presented here indicate that MAR-BP1 and NF-µNR are distinct. Complexes formed by NF-µNR and DNA containing its recognition sites form characteristic complexes that migrate very slowly in MSA gels. Complexes with MAR-BP1 migrate more rapidly. NF-µNR binding generates complexes that are easily analyzed by DNase I footprinting, whereas MAR-BP1 complexes are not. The most convincing argument that they are distinct is that UV-cross-linking reveals proteins of different sizes.
MAR-BP1 also
appears to be distinct from histone H1. Purified histone H1 from the
same B cell line has different DNA-binding/competition characteristic
and antigenicity as compared with MAR-BP1. ()
Our results describe the identification of a novel MAR-binding protein that specifically binds to MAR sites associated with the IgH intronic enhancer. Although we cannot rule out the possibility that other matrix proteins are involved in the association between the nuclear matrix and the IgH enhancer in B cells, MAR-BP1 shows the strongest MAR-binding activity in our soluble nuclear matrix preparation. In addition, MAR-BP1 seems to be a general MAR-binding protein since the competition experiments presented in Fig. 2indicate that it interacts with a number of other MAR regions.
An alternative
hypothesis for MAR/matrix interaction has been proposed based on the
observation that the MARs flanking the IgH enhancer assume a stable
unpaired conformation at high superhelical density, that extensive
base-unpairing is important for matrix attachment (Kohwi-Shigematsu and
Kowhi, 1990). In support of this idea, mutations within NF-µNR site
P3 that abolish base-unpairing also adversely affected matrix binding in vitro (Bode et al. 1992). These results imply that
MAR-binding proteins would show preferential interaction with
single-stranded DNA. However, while MAR-BP1 binding can be competed by
double-stranded P4 DNA, it was unaffected by either single-stranded P4
oligonucleotide.
It now appears that MAR-binding proteins can be divided into two groups. One group included proteins like nucleolin (Dickinson and Kowhi-Shigematsu, 1995) and Lamin B (Hakes and Berezney, 1991), which have a propensity to bind single-stranded DNA. The other group includes SATB1 and MAR-BP1, which bind double-stranded DNA, possibly through the recognition of a particular tertiary structure such as a narrow minor groove.
The
binding sites for several MAR-binding proteins differ from the
recognition sites of nuclear transcription factors in that a relative
large fragment (200 bp) is required for high affinity binding (von
Kries et al., 1991, 1994; Tsutsui et al., 1993;
Luderus et al., 1994). In the case of MAR-BP1, this would
explain why removal of the XbaI-RsaI region from the
5`-end of the 5`-En fragment reduces binding affinity by a factor of 4
and yet has no intrinsic affinity for MAR-BP1 itself (Fig. 5).
In any case, it is clear that deletion of either P1 or P2 on the 5`-En fragment, or P3 or P4 on the 3`-En fragment had profound effects on MAR-BP1 binding suggesting a requirement for cooperative interactions. It is possible that these sequences serve as nucleation sites for a more extensive covering of the region by multiple MAR-BP1 proteins. This could explain why it has proven difficult to footprint these, or any, DNA regions in MAR-BP1 complexes.
Taken together, these results provide support for a model of NF-µNR-mediated enhancer regulation involving nuclear matrix attachment previously proposed (Scheuermann and Chen, 1989). In this model (see Fig. 9) an important aspect of IgH enhancer function would be to attach the heavy chain locus to the nuclear matrix in appropriate cells (B lymphocytes). This would bring the gene into regions of the nucleus that contain high concentrations of transcription factors, RNA polymerase, and topoisomerases. However, in inappropriate cells (other than B lymphocytes) NF-µNR would be expressed and would bind to its recognition sites flanking the enhancer, thereby preventing nuclear matrix attachment.
Figure 9: A model for the regulation of IgH enhancer activity through differential association with the nuclear matrix. Transcription in interphase nuclei is apparently organized into a limited number of focal points that can be visualized by immunofluorescence (Jackson et al., 1993). These focal points contain elongating RNA chains, RNA polymerase, and other components of the transcription apparatus. These focal points are bound to a matrix present within the nucleus, perhaps at areas of intersection between nuclear matrix filaments. DNA is bound to these focal regions through the action of MAR-binding proteins (triangles) like MAR-BP1. In B cells, where the IgH locus is transcriptionally active, the MARs flanking the IgH enhancer are bound to these foci facilitating the interaction with transcriptional components. In non-B cells, where the locus is silent, the MAR sites flanking the enhancer are bound by NF-µNR (spheres labeled N) preventing the IgH locus from associating with the focal points of high transcriptional activity.
This model implies that MARs are transcriptional regulatory elements. The first indications that MARs might be involved in transcription came from the observation that MARs are frequently found near cis-acting transcriptional regulatory elements (e.g. Gasser and Laemmli, 1986; Cockerill and Garrard, 1986; Cockerill et al., 1987). Analysis of the effects of MARs on transcriptional activity has revealed an interesting characteristic of MARs that distinguish them from promoter and enhancer elements. MARs have been found to stimulate transcription from defined promoters in a number of systems, but only when the test construct is stably integrated into a chromosomal context (e.g. Blasquez et al., 1989; Xu et al., 1989; Klehr et al., 1990). In addition to increasing the level of transcription, MARs seem to provide for copy number-dependent and position independent transcription (Phi-Van et al., 1990; McKnight et al., 1992). Although locus control regions can have similar effects, it is not clear whether they are the same as MARs or similar (Dillon and Grosveld, 1993).
Some evidence suggests that
MARs might play a role in tissue-specific expression regulation. Genes
that are actively transcribed are preferentially associated with the
nuclear matrix, while genes that are not expressed are not bound
(Gerdes et al., 1994). In addition, it has been found that the
-globin gene is associated with the nuclear matrix in
reticulocytes but not in thymocytes, whereas the malic enzyme gene is
attached in thymocytes but not reticulocytes (Brotherton et
al., 1991). This observation demonstrates that tissue-specific
nuclear matrix attachment correlates with tissue-specific
transcriptional expression.
In the case of the IgH intronic enhancer, a recent publication provides strong support for the importance of these MAR regions in transcriptional enhancement (Forrester et al., 1994). The ability of the IgH enhancer to stimulate expression of a rearranged Ig heavy chain transgene was examined in preB cell lines isolated from transgenic mice in which the transgene contained a wild-type enhancer or an enhancer with MAR site deletions. While all preB cell lines from mice containing the wild-type enhancer exhibited high level transgene expression, transgene expression was absent in lines from MAR deletion mutants. These results indicate that, in mice, IgH enhancer activity requires intact MARs. As NF-µNR is only expressed in non-B cells and can prevent nuclear matrix attachment in vitro, the cell-type-specificity of the IgH enhancer may in part be controlled by the negative regulation of nuclear matrix attachment.