MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 5206-5211 0270-7306/91/105206-06$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 10

Identification of a Matrix-Associated Region 5' of an Immunoglobulin Heavy Chain Variable Region Gene CAROL F. WEBB,'* CHHAYA DAS,2 KENTON L. ENEFF,' AND PHILIP W. TUCKER2 Department of Immunobiology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104,' and Department of Microbiology, Southwestern Medical Center at Dallas, Dallas, Texas 7310442 Received 13 February 1991/Accepted 24 July 1991

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the IL immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A+T/G+C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.

Gene expression, in general, is effected by chromosomal position and may reflect the organization of DNA into topologically constrained loops that act as functional domains (reviewed in reference 11). These loops are thought to be organized by anchoring of the DNA to the nuclear scaffold or matrix and may function to associate transcriptionally active regulatory regions that would otherwise be far apart. The nuclear matrix is the proteinaceous nuclear subfraction that remains after histone depletion and forms a scaffold for chromosome attachment (reviewed in reference 11). Matrix-associated regions (MARs) have been functionally defined as the A+T-rich DNA sequences that are preferentially retained by this chromosomal framework (6, 12). MARs are usually 200 to 300 bases long, typically contain topoisomerase II cleavage sites, and occur on the average of every 30 kb in eucaryotic DNA (1, 11, 19, 21). While the presence of these sequences has been highly conserved during evolution such that DNA from one species can bind to nuclear matrix proteins from diverse cell types, the MAR sequences themselves do not generally share adequate homology to cross hybridize (2, 11, 15). Furthermore, despite the ability of MAR sequences to bind to matrix proteins from diverse cell types, a large number of nuclear matrix proteins (61%) may be expressed in a cell-typespecific manner (10). The importance of 5'-flanking sequences and the location of MARs near enhancer and promoter regions of the human ,-globin and chicken lysozyme genes have recently become apparent (22, 23). Three developmentally regulated Drosophila genes have also been shown to be bounded by both 5' and 3' nuclear scaffold attachment regions (12). A direct relationship between MARs and gene expression was previously noted within the immunoglobulin (Ig) K light chain locus (6). Deletions of the MARs which flank the enhancer within the VJ-CK intron decreased K expression, implying

intronic and is flanked by MARs that may also be important for heavy chain transcription (7). While positive regulatory proteins have been demonstrated to bind near these regions in B cells, putative negative regulatory proteins appear to bind these regions in nonlymphoid cells (14, 20). Whether proteins may function both as MAR components and as transcriptional activators or repressors is not known; however, the cell type specificity of a large number of the MAR proteins (10) suggests that it is a likely possibility. One nuclear scaffold protein (RAP-1) has been purified from yeast cells (13). It mediates the formation of DNA loops in vitro and may play a role in silencing the HML mating type locus. More recently, a protein that binds to the MAR in the chicken lysozyme locus has been purified and may be important for establishing active domains at this locus (24). In the accompanying report, we used an antigen-specific transfected cell line to demonstrate that the cytokine interleukin-5 (IL-5) plus the antigen (Ag) phosphocholine-conjugated keyhole limpet hemocyanin induced increased abundances of two protein-DNA complexes (26). One of these complexes (VTXE) bound to sequences between -214 and -160 relative to the transcription start site of the transfected ,. heavy chain gene. These sequences were required for the observed increases in Ig transcription in cells treated with IL-5 plus Ag. The other IL-5-plus-Ag-inducible proteinDNA complex (VDSE) bound to sequences between -525 and -462 and was shown to be similar to the more 3' complex by both protein cross-linking and competition experiments. Both of these sequences were >72% A+T rich, but there was little direct sequence homology between the two binding sites. In an attempt to further characterize the functions of these protein-DNA complexes and because the DNA sequences were also very A+T rich, we examined their ability to bind to the nuclear matrix. Our data clearly demonstrate that DNA fragments containing the more 5' of these binding sites can act as a MAR in vitro. Furthermore, protein crosslinking studies have identified a 40-kDa matrix protein that binds to the same region bound by IL-5-plus-Ag-inducible nuclear extract proteins.

that the MARs themselves may play a positive role in transcription (3). The Ig ,u heavy chain enhancer is also *

Corresponding author. 5206

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FIG. 1. Protein-DNA interactions induced by IL-5 plus Ag. A diagram of the heavy chain Ig locus shows the orientations of known enhancer sequences (solid diamonds), octamer (solid square), and heptamer (solid circle), protein-binding sites, and sequences known to act as MARs (M). The IL-5-plus-Ag-inducible protein-binding sites VDSE (triangle) and VTXE (open diamond) are also indicated. The S107 V-region gene and leader sequences are depicted by open and asterisked bars, while the CR genes are indicated by vertical stripes. An arrow shows the transcription start site. The promoter-containing 550-bp fragment and the bflSO and TX125 fragments are indicated below.

MATERIALS AND METHODS

Cell culture. The BCg3R-ld transfected cell line is a derivative of the BCL1Bj murine B-cell lymphoma and has been described previously (25). It contains genomic sequences encoding ,u and K Ig chains with S107 variable (V) regions, and Ig produced by these cells binds phosphocholine. Growth medium consisted of RPMI 1640 with 10% fetal calf serum, 5 x 1O-5 M P-mercaptoethanol, 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, and 10 mM glutamine and sodium pyruvate. Stimulation with 0.5 ng of recombinant IL-5 per ml and 50 ng of the antigen phosphocholine-conjugated keyhole limpet hemocyanin per ml was performed for 20 to 24 h as previously described (25). IL-5 was graciously provided by R. L. Coffman (DNAX, Palo Alto, Calif.). Nuclear extract and matrix preparation. Nuclear extract proteins were prepared by hypotonic lysis of isolated nuclei as previously described (18). Soluble proteins were dialyzed, and protein concentrations were measured by the Bradford assay (Bio-Rad Laboratories, Richmond, Calif.). For nuclear matrix preparation, nuclei were additionally purified by centrifugation through a 2 M sucrose cushion, and matrices were isolated as previously described (6). Briefly, isolated nuclei were digested with 100 ,ug of DNase per ml for 2 h at room temperature and were extracted three times with 2 M NaCl-10 mM EDTA-10 mM Tris-HCl-0.5 mM phenylmethylsulfonyl fluoride-0.25 mg of bovine serum albumin (BSA) per ml. The resulting insoluble nuclear matrices were pelleted, washed in 10 mM NaCl-3 mM MgCI2-10 mM Tris-HCl (pH 7.4)-0.25 M sucrose-0.25 mg of BSA per ml, and resuspended in the same solution with an equal volume of glycerol before storage at -70°C. Nuclear matrix-binding assay. Binding to the nuclear matrix was measured exactly as described elsewhere (6). Briefly, matrices were washed three times in 50 mM NaCl-10 mM Tris-HCl (pH 7.4)-i mM MgCl2-0.25 M sucrose-0.25 mg of BSA per ml and were incubated at 23°C with 20 ng of 32P-end-labeled DNA fragments per ml and 10 ,ug of Escherichia coli carrier DNA per ml for 2 h. Competition experiments were performed by adding either unlabeled fragment or nuclear extract proteins to the samples at the beginning of the incubation. After washing away DNA that was not bound to the insoluble matrix pellet, proteinase K was added to digest the protein structure, and matrixbound DNA fragments were purified by phenol extraction and precipitation. These fragments were electrophoresed on 7.5% polyacrylamide gels, dried, and autoradiographed. The DNA probes used were a 150-bp BamHI-FokI frag-

ment (bflS0) spanning the sequence from -574 to -425 relative to the transcription start site of the S107 heavy chain genes, a similar fragment from the same area spanning bases -251 to -124 (TX125), and a 574-bp BamHI fragment containing both previously described sequences and all of the remaining sequence from -574 to the transcription start site. All fragments were cloned into pUC19, and the entire plasmid was digested with the appropriate enzyme and end labeled. Fifty percent of the input DNA originally incubated with the matrix was used as a standard to show specificity of matrix binding to cloned fragments versus the vector DNA. UV cross-linking proteins to DNA. Cross-linking of nuclear extract proteins to DNA by UV irradiation was carried out as described elsewhere (4). Briefly, oligomers prepared to the 46-bp nuclear-extract-binding sites of the bflS0 fragment defined elsewhere (26) were incubated with 5 ,ug of extract for 20 min at 37°C and were subjected to UV irradiation for 45 min. The oligomer complimentary to the region from -515 to -476 was labeled on the coding strand such that [_y32P]dATP and Br-dUTP replaced dATP and dTTP in the sequence 5' CTTGT'TT7AYTAACTTATITTATCTTA 3'. After cross-linking, the samples were digested with 40 U of DNase I and 1.0 U of micrococcal nuclease for 30 min at 37°C. Competition experiments were performed by adding unlabeled fragment, or a nonspecific octamer motif-containing oligomer (18), to the samples 5 min prior to the addition of the labeled fragment. Samples were electrophoresed at 60 mA in 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels under reducing conditions, and the gels were dried and autoradiographed at -70°C. Soluble nuclear extract proteins were used directly from stored aliquots. Nuclear matrix proteins were solubilized either before or after cross-linking in 65 mM dithiothreitol-8 M urea-2% Nonidet P-40-65 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) as

previously described (8). RESULTS Sequences 5' of the Ig promoter bind to the nuclear matrix and act as MARs. In the accompanying paper, we demonstrate by mobility shift assay that IL-5 plus Ag induces increased abundances of nuclear extract proteins that bound to two A+T-rich sequences (bflS0 and TX125) 5' of the basal Ig promoter (26). A diagram summarizing these findings and other known regulatory regions of the p. heavy chain Ig locus is shown in Fig. 1. Because MARs have been identified as A+T-rich DNA sequences that often occur in close proximity to regulatory sequences (reviewed in reference 11), we asked whether our sequences were capable of direct binding

5208

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1 23 4 5 6 78 b FIG. 2. Binding to the nuclear matrix. Sequences were examined for their ability to bind to the nuclear matrix. (a) Nuclear matrix preparations were incubated with whole 5'-end-labeled BamHIdigested plasmids containing either the 550-bp BamHI fragment with the entire VH promoter region to -574 (lanes 1 to 4) or the bf150 fragment containing sequences from -574 to -425 (lanes 6 to 8). The matrix-retained fragments obtained after washing away the unbound DNA and digesting the matrix proteins with proteinase K are shown in lanes 2 to 4 and 6 to 8. Lanes 1 and 5 contain 50% of the free input DNA added to the matrix, and the unmarked bands represent labeled plasmid DNA. Lanes 3 and 7 also contained a 100-fold molar excess of unlabeled bflS0 as a competitor, while lanes 4 and 8 contained a 1,000-fold molar excess of bflS0. (b) Nuclear matrix preparations were incubated with a 3'-end-labeled HaeII-digested plasmid that contained the entire TX125 fragment within a 570-bp HaeII fragment. In the right lane, a 100-fold molar excess of the unlabeled bflSO fragment was added to the reaction as a competitor for binding.

to the nuclear matrix. Therefore, nuclear matrices from the IL-5-plus-Ag-inducible B-cell transfectant BCg3R-ld were examined for their ability to specifically retain DNA fragments containing these sequences. Figure 2 shows that the 550-bp BamHI fragment (shown in Fig. 1) containing both IL-S-plus-Ag-inducible regions and the entire promoter region acted as a very strong MAR. Likewise, the 170-bp bflS0 fragment alone was a strong MAR, even though it is a shorter fragment than is generally thought to be necessary for matrix association. Binding of both fragments could be specifically inhibited by a 100-fold molar excess of the unlabeled bflS0. The control vector fragments were not specifically retained by the protein framework in any case. We were unable to demonstrate matrix binding with the more promoter-proximal 125-bp TX125 fragment in any of six experiments (data not shown). Failure to bind to the matrix could not be explained by insufficient fragment length alone, since a 570-bp HaeII fragment containing the entire TX125 sequence and additional sequences from pUC19 was not retained by the matrix (Fig. 2b). Thus, the bflSO fragment can function as a MAR, while the TX125 fragment did not. To determine whether the bflSO fragment was binding to the nuclear matrix through cell-type-specific interactions, we examined the ability of this fragment to be bound by nuclear matrices prepared from the T-cell line EL-4. Nuclear extract proteins from B-cell lines exhibited B-cell-specific protein interactions with this fragment by mobility shift assay,

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FIG. 3. Matrix binding is not cell type specific. Nuclear matrix proteins from both B- and T-cell lines bound to the bflSO fragment. A BamHI-digested, end-labeled plasmid containing the bflSO fragment was incubated with matrix proteins obtained from the T-cell line EL-4 or the B-cell line BCg3R-ld. The 170-bp fragment is labeled, and an arrow designates the single-stranded form of the fragment. INPUT lanes contain 50% of the free input counts added to the matrix proteins; the unlabeled bands in these lanes represent plasmid DNA.

although T-cell extracts do contain proteins of a distinct mobility that can bind to these sequences (26). Figure 3 shows that both B- and T-cell-derived matrices were capable of binding to the bflS0 fragment. No quantitative differences in binding were observed consistently. In some cases, a portion of the retained fragment electrophoresed at an aberrantly slow mobility that was identical to the single-stranded form of the fragment, as indicated by the arrow. This is probably an artifact that may result from the high A+T/G+C ratio of the fragment, since single-stranded DNA could also be observed in the protein-free, input DNA. Likewise, no differences in the ability of fragments to be retained by matrices were observed when matrices were prepared from either IL-S-plus-Ag-induced or uninduced BCg3R-ld cells (data not shown). Thus, as previously seen by Cockerill and Garrard (6) for the CK MAR, the transcriptional status of our region had no consistent quantitative or qualitative effects on MAR binding. Soluble proteins in nuclear extracts block binding to the nuclear matrix. A critical issue raised by the matrix data is whether the same sequences that are bound by the VDSE proteins in a mobility shift assay using soluble nuclear extracts are also involved in binding to proteins present in the matrix. To address this, we preincubated fragments with induced or uninduced nuclear extracts first, then added matrix proteins to the samples, and assayed the effects on MAR binding. Both uninduced and induced extracts consistently competed for binding to matrix proteins. The results using uninduced extracts are shown in Fig. 4. The simplest interpretation is that proteins present in the extracts protected DNA critical to the MAR interaction. This may not be surprising, since both the gel-shifted complex and the nuclear matrix seem to interact with fairly long sequences. Nuclear matrix proteins bind to the same sequences protected by nuclear extract proteins. The VDSE protein-binding site for nuclear eXtract proteins was identified elsewhere as a 46-bp sequence within the bflS0 fragment (26). Attempts to compete for matrix binding with an oligomer to this binding

MATRIX ASSOCIATION REGION 5' OF Ig LOCUS

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site were unsuccessful (data not shown). However, matrices do not bind effectively to fragments shorter than 150 to 200 bp (11), and differences in binding affinity might obscure specific interactions with shorter sequences. To test whether matrix proteins interacted directly with sequences within the 46-bp oligomer protected by IL-5-plusAg-inducible proteins, UV cross-linking studies were per-

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FIG. 6. Hypothetical model for role of the matrix in Ig transcription. A schematic drawing of the Ig locus including the variable (V1) and constant (C,u) gene segments is shown. The arrow denotes the transcription start site, while the open squares, open diamond, and open circles depict the octamer, heptamer, and pyrimidine regulatory motifs, respectively, that have been previously identified 5' of the Ig locus (9). The shaded symbols represent similar motifs which have been identified with in the intronic enhancer region. The shaded rectangles represent DNA sequences that may act as matrix attachment regions. The next closest MAR to the 5' and intronic, enhancer-associated MARs is between CB and C-Y3 about 40 to 50 kb downstream (5). Hypothetical configurations of the regulatory regions relative to the nuclear matrix are shown for both silent genes and for genes that are undergoing initiation of transcription. Jagged lines represent the nuclear matrix.

have demonstrated the existence of a MAR approximately 500 bp 5' of the basal Ig promoter. Matrix proteins in both yeast and chicken cells have been shown to participate in the formation of looped DNA domains (13, 24). The identification of a 5' MAR defines a looped domain containing the Ig heavy chain promoter and intronic enhancer regulatory sequences. The basis by which the intronic enhancer affects Ig transcription from a distance of 2.6 kb is not clear. Speculations have been made that these elements interact through common binding proteins. The S107 VH promoter and IgH enhancer share, at a minimum, three DNA-binding motifs (octa, ,uE3, and ,uEPB-E) (9), two of which (,uEPB-E and octa) are transcriptionally functional in both the promoter and enhancer regions (17). Our data provide a topological model for these interactions through the formation of chromosome loops which could bring these distally located elements into close proximity through binding to the nuclear matrix (Fig. 6). Thus, matrix proteins may facilitate interactions between enhancer- and promoter-associated regulatory proteins during the initiation of Ig transcription. Matrix proteins and IL-5-plus-Ag-inducible proteins bind to the same site. Our UV cross-linking studies indicated that the same sequences that bound to IL-S-plus-Ag-inducible proteins in another study (26) also interacted with matrix proteins. Other examples in which regulatory regions have been found in close proximity to MARs have been well documented (6, 12, 22, 23). Whether some of the same proteins are involved in the B-cell-specific binding to VDSE after IL-5-plus-Ag induction and in matrix attachment is unknown. Although the binding complexes that we detected in mobility shift assays exhibited cell-type-specific mobilities, the major components of the nuclear matrix (i.e., topoisomerase II, lamins, and ribonucleoproteins) are present in all cell types. Matrices from T cells exhibited indistinguishable binding to the bfl50 fragment. Nor was binding affected when matrices were isolated from IL-5-plus-

Ag-induced rather than uninduced cells. While the apparent B-cell specificity of the induced protein-DNA complex might seem to rule out binding of these proteins to the matrix, a large number of cell-type-specific proteins have been demonstrated to be present in very low levels in nuclear matrices (10). However, cell-type-specific proteins did not appear to be required for matrix binding to bflS0. On the other hand, the proteins bound in mobility shift assays may be distinct from those that participate in MAR binding. Scheuermann and Chen (20) have proposed that a protein that binds to the Ig enhancer and acts as a negative regulator of transcription may interfere with MAR binding. In our system, however, IL-5-plus-Ag-induced protein binding is correlated with positive regulation of transcription. Our previous studies indicated that the mobility-shifted proteins bound to both the TX125 and bflS0 sequences were very similar both by competition experiments and by UV cross-linking. However, only the bflS0 fragment acted as a MAR in this study. Thus, the matrix proteins that bind to bflS0 may be distinct from the IL-5-plus-Ag-inducible proteins that bind to the same sequences, despite the apparent similarity in molecular mass (about 40 kDa) of one of the inducible proteins and the matrix protein. Alternatively, the IL-5-plus-Ag-inducible mobility-shifted complexes that we observed elsewhere (26) are probably composed of more than one protein, and some of these components could be shared by different cell types. Thus, these ubiquitous proteins might participate in matrix attachment, while interactions with cell-type-specific proteins could account for differences in the mobility-shifted complexes. An answer to the important question of whether any of the proteins that produce mobility shifts are the same proteins that participate in MAR interactions must await protein purification. Relationship to other MAR-binding proteins. Although at least two other MAR-binding proteins have already been

VOL . 1 l, 1991

characterized, both Rap-1 in yeast cells and the ARBP protein from chicken cells have apparent molecular masses in excess of 90 kDa (13, 24). Although we can not precisely determine the contribution that the undigested, cross-linked DNA makes to the molecular weight of our protein, it is clearly smaller than either Rap-1 or ARBP. Thus, it may represent a novel matrix protein. The ability of the matrix protein to retain its ability to bind to DNA in the presence of 8 M urea is precedented. At least one other protein, a heat shock protein from Drosophila cells, has been shown to retain sequence-specific binding activity in the presence of 8 M urea (16). This protein also binds to sequences that are known to contain a scaffold attachment region and may be associated with the nuclear matrix. In any case, the ability to retain DNA-binding activity in the presence of high concentrations of urea may aid in its purification. The bflSO binding site contained a repeat of the 12-bp sequence AACTTGTTITATT. This sequence was found in the 5'-flanking and intronic regions of several genes from many different species (26). Homologies were located near known enhancer or promoter regions in many cases, and in several cases, chromosomal attachment regions 5' of the homologous sequence have been shown to influence gene activity (22, 23). Therefore, the matrix proteins that bind to the bflSO sequences may play important roles in other gene systems as well. ACKNOWLEDGMENTS We thank Robert Coffman for providing IL-5 and William Garrard for helpful discussions. This work was supported by NIH grants CA31534 and A118016 to P.W.T. REFERENCES 1. Adachi, Y., E. Kas, and U. K. Laemmli. 1989. Preferential, cooperative binding of DNA topoisomerase II to scaffoldassociated regions. EMBO J. 8:3997-4006. 2. Amati, B. V., and S. M. Gasser. 1988. Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold. Cell 54:967-978. 3. Blasquez, V. C., M. Xu, S. C. Moses, and W. T. Garrard. 1989. Immunoglobulin K gene expression after stable integration. I. Role of the intronic MAR and enhancer in plasmacytoma cells. J. Biol. Chem. 264:21183-21189. 4. Chodosh, L. A., R. W. Carthew, and P. Sharp. 1986. A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol. Cell. Biol. 6:4723-4733. 5. Cockerill, P. N. 1990. Nuclear matrix attachment occurs in several regions of the IgH locus. Nucleic Acids Res. 18:26432648. 6. Cockerill, P. N., and W. T. Garrard. 1986. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44:273-282. 7. Cockerill, P. N., M.-H. Yuen, and W. T. Garrard. 1987. The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements. J. Biol. Chem. 262:5394-5397. 8. Cupo, J. F., P. Lidgard, and W. F. Lichtman. 1990. A high resolution two-dimensional gel electrophoresis and silver stain-

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