Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10526-10530, November 1995 Cell Biology

The nuclear matrix NMP-1 is the factor YY1 ( expression/histone H4/nuclear structure/nucleolus/osteoblast) Bo Guo*, PAUL R. ODGREN*, ANDREI J. VAN WIJNEN*, THOMAS J. LAST*, JEFFREY NICKERSONt, SHELDON PENMANt, JANE B. LIAN*, JANET L. STEIN*, AND GARY S. STEIN* *Department of Cell Biology and Cancer Center, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655; and tDepartment of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 Contributed by Sheldon Penman, August 7, 1995

ABSTRACT NMP-1 was initially identified as a nuclear have been associated with MARs, such as topoisomerase II matrix-associated DNA-binding factor that exhibits sequence- cleavage sites and several short A+T-rich sequences. Using specific recognition for the site IV regulatory element of a chemical protection assays with solubilized nuclear matrix histone H4 gene. This distal domain is a nuclear , we have previously characterized a nuclear matrix- matrix interaction site. In the present study, we show that associated protein, NMP-1, which recognizes a sequence motif NMP-1 is the multifunctional YY1. Gel- within the site IV promoter element (31). Prerequisite to shift and Western blot analyses demonstrate that NMP-1 is understanding the function of NMP-1 in histone H4 gene- immunoreactive with YY1 antibody. Furthermore, purified nuclear matrix interactions and transcriptional control is iden- YY1 protein specifically recognizes site IV and reconstitutes tification of NMP-1. the NMP-1 complex. Western blot and gel-shift analyses YY1 (also known as 6, NF-E1, or UCRBP) is a ubiquitous indicate that YY1 is present within the nuclear matrix. In situ transcription factor that has been shown to interact with immunofluorescence studies show that a significant fraction regulatory sequences for a number of mammalian , both of YY1 is localized in the nuclear matrix, principally but not cellular and viral (reviewed in refs. 32 and 33). An intriguing exclusively associated with residual nucleoli. Our results property of YY1 is that it can either activate or repress confirm that NMP-1/YY1 is a ubiquitous protein that is transcription or be functionally neutral, depending on the present in both human cells and in rat osteosarcoma ROS promoter context in which it occurs. Several studies have 17/2.8 cells. The finding that NMP-1 is identical to YY1 indicated that association with other proteins can significantly suggests that this transcriptional regulator may mediate modulate the activity of YY1 (34-36); for example, Shrivas- gene-matrix interactions. Our results are consistent with the tava et al. (35) have shown that c- inhibits both the concept that the nuclear matrix may functionally compart- repressor and activator functions of YY1. In the c-fos promoter mentalize the eukaryotic nucleus to support regulation ofgene YY1 appears to act by bending DNA to regulate contact expression. between other factors (37). Thus, YY1 has been implicated in several possible functional roles related to transcription. The nuclear matrix has been linked to multiple functions that In this study, we unequivocally establish that the nuclear mediate the control of within the nucleus matrix protein NMP-1 is the transcriptional regulator YY1 and (1-8). This component of the nuclear architecture provides the demonstrate that NMP-1/YY1 is localized to the nuclear internal scaffold of the nucleus; it consists of a peripheral matrix in situ as well as by biochemical analyses. lamina-pore complex, an internal filamentous ribonucleopro- tein network, and residual nucleoli (2, 8). The nuclear matrix MATERIALS AND METHODS plays an important role in higher-order organization of chro- matin and the tethering of actively transcribed genes via Nuclear Protein Preparations. Nuclear extracts were pre- matrix-associated regions (MARs) or scaffold-attached re- pared as described (38) by 0.42 M KCl salt extraction of nuclei gions (SARs) (9-18). Involvement of the nuclear matrix in from HeLa S3 cells grown in suspension culture as well as of RNA processing has been demonstrated; heterogeneous nu- nuclei from ROS 17/2.8 cells grown in monolayer cultures. clear RNAs, small nuclear RNAs, and RNA processing inter- Nuclear matrix proteins were obtained by solubilization of mediates are enriched in the nuclear matrix; ribonucleoprotein nuclear matrices and removal of intermediate filament pro- particles are components of nuclear matrix structure (19-22). teins as described (2). The presence of ubiquitous and tissue-specific transcription Synthesis of YY1. Recombinant YY1 protein was obtained factors (23-25), as well as steroid hormone receptors and by overexpression in the Escherichia coli K-12-derived corresponding acceptor proteins (26-29), in the nuclear matrix M15[pREP4] strain transformed with a His/YY1 plasmid of many distinct cell types and tissues supports a role for the (generously provided by E. Seto, University of Texas, San nuclear matrix in transcriptional regulation of gene expression. Antonio). YY1 expression was induced by treatment of bac- Exploring mechanisms that mediate interactions between terial cultures with 1 mM isopropyl 3-D-thiogalactopyranoside DNA and structural protein components in the nuclear matrix for 4 hr. His/YY1 was purified from bacterial lysates by nickel requires analysis of the proteins and DNA sequences involved chelate column chromatography (34). in the association of actively transcribed genes with the nuclear Gel-Shift Assays. Nuclear protein or bacterially expressed matrix. Recently, we have identified a distal promoter element, protein (1 ,ug) was added to 20-,l reaction mixtures in binding site IV, in a human histone H4 gene promoter that resides buffer containing 12.5 mM Hepes (pH 7.5), 50 mM KCl, 10% within a sequence domain that has properties of a MAR (30, (vol/vol) glycerol, 1 mM dithiothreitol, 0.2 mM EDTA, 1 ,ug 31). This domain preferentially associates with nonhistone of poly(dI-dC)-(dI-dC) and incubated with 20 fmol of 32P-end- chromosomal proteins and contains consensus sequences that labeled double-stranded oligonucleotides at room temperature for 15 min. Competition assays were performed by inclusion of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: MAR, matrix-associated region; Rb, retinoblastoma accordance with 18 U.S.C. §1734 solely to indicate this fact. gene product.

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H4 Promoter H4 mRNA to verify chromatin removal. The distribution of antigens was indistinguishable in single antigen and colocalization experiments site IV sitellil site site 1I as well as in different cell lines.

NMP-1 RESULTS -730 -589 The Nuclear Matrix Protein NMP-1 Is Identical to Tran- scription Factor YY1. Previous studies have indicated that a GATTCGCTGACGTCCATGAGAAAGCTT 141-bp fragment in the distal promoter (site IV, -730 to CTAAGCGACTGCAGGTACTCTTTCGAA -589) of the human histone H4 gene preferentially associates -651 * -625 with the nuclear matrix (31). Site IV interacts with a sequence- YY-1 specific DNA binding activity, designated NMP-1, isolated nuclear matrix (ref. 31; Fig. 1). FIG. 1. Schematic representation of the NMP-1 binding site in the from solubilized preparations The NMP-1 recognition site contains both an asymmetrical histone H4 gene. The fragment between nucleotides -730 and -589 was initially identified by in vitro mapping of nuclear MARs. The ATF/CREB consensus motif (TGACGTCC) and a binding site NMP-1 binding site was delineated by deletion analyses and 1,10- for the transcriptional regulatory YY1 (CGTCCATG; CCAT phenanthroline-copper chelate protection assays. Specific contacts of core underlined) (32). Previous results from UV-crosslinking and NMP-1 with guanines as established by methylation interference mutational analysis suggested that members of the ATF family analysis (31) are indicated by asterisks. YY1 consensus motif is might be involved in NMP-1 complex formation (31). bracketed. To investigate the protein components of NMP-1, we de- signed three mutant site IV oligonucleotides (Table 1) in which of unlabeled oligonucleotides unless indi- 500 fmol (25 nM) the NMP-1 binding site TGACGTCCATG was changed to cated otherwise. For supershift experiments, 1 ,ug of antibody TGAtcTCCATG (mutant 1), TcAgcaCCATG (mutant 2), or was added to protein mixtures and incubated on ice for 30 min TGACGTCagaG (mutant 3). These oligonucleotides, as well prior to addition of probe. Reaction mixtures were electro- as oligonucleotides containing consensus binding sites for the phoretically fractionated in a 4% nondenaturing acrylamide ATF family or for the related AP1 family of transcription gel at 200 V for 2 hr. Gels were dried and subjected to factors, were used in a series of gel-shift competition assays in autoradiography with Kodak XAR film. which the wild-type site IV oligonucleotide was used as probe Immunoblotting. SDS/PAGE, electroblotting, and immu- (Fig. 2). Neither the consensus palindromic ATF nor consen- nostaining (ECL, Western blotting protocols; Amersham) sus AP1 oligonucleotide was able to compete for NMP-1 were carried out as described (39). Southwestern blotting was binding to site IV (Fig. 2A). In addition, both mutants 1 and performed as reported (40). 2, which alter nucleotides that are essential for ATF binding as Immunofluorescent Localization. HeLa (S3 or CCL2) or well as represent key recognition base pairs for NMP-1, CaSki cells were grown on coverslips. Soluble and chromatin showed only limited competition for NMP-1 binding (Fig. 2B). proteins were removed by a modification of the protocol of Fey In contrast, mutant 3, which has an intact ATF binding site, has et al. (2). Soluble proteins were extracted with 0.5% Triton completely lost the ability to compete for NMP-1 binding (Fig. X-100 in cytoskeletal buffer before chromatin removal by 2B). Taken together, these results support the conclusion that DNase I digestion (600 units/ml for 1 hr at 32°C) and 0.25 M the ATF core motif is not a primary determinant for interac- ammonium sulfate extraction. Vanadyl ribonucleoside com- tion of NMP-1 with site IV. Thus, NMP-1 is not a member of plex (20 mM) and 4-(2-aminoethyl)benzenesulfonyl fluoride (1 the ATF/CREB family of transcription factors. mM) were present in all solutions. Nuclear matrices were fixed As noted above, the NMP-1 binding site contains a potential with 4% formaldehyde in cytoskeletal buffer (2) for 40 min at 4°C, interaction sequence for the transcriptional regulator YY1 washed with phosphate-buffered saline (PBS), and blocked with (5'-CGGCCATCTT-3'; underlined sequences represent nu- 10% normal goat serum in PBS. Antibody incubations were cleotides matching site IV). The CCAT core sequence is the performed in PBS containing 0.1% Tween 20 and 1% normal principal determinant for YY1 binding (32). Mutant 3, which goat serum. Primary antibodies were rabbit anti-YY1 (Santa does not compete with NMP-1 binding to the site IV probe Cruz Biotechnology), a monoclonal antibody against Numatrin/ (Fig. 2B), contains a substitution within the CCAT core motif. B23 (kindly provided by P. K Chan, Baylor College of Medicine, Therefore, NMP-1 may be YY1 related. To assess whether Houston), and monoclonal antibodies against lamin B and NuMa NMP-1 represents a DNA binding activity that is related to (provided by Matritech, Cambridge, MA). Second antibodies YY1, we performed oligonucleotide competition and antibody were goat anti-mouse IgG coupled to either rhodamine or supershift experiments with nuclear matrix protein from both fluorescein. In single labeling experiments, cells were counter- HeLa (Fig. 3A) and ROS 17/2.8 (Fig. 3B) cells in gel mobility- stained with 4',6-diamidino-2-phenylindole (5 ,ug/ml for 5 min) shift assays. The competition results demonstrate that NMP-1 Table 1. Sequences of oligonucleotides used for gel-shift and competition assays NMP-1 binding Oligonucleotide Sequence activity Site IV GATTCGCTGACGTCCATGAGAAAGCTT +++ ATF consensus AGAGATTGCCTGACGTCAGAGAGCTAG AP1 consensus CGCTTGATGACTCAGCCGGAA Site IV mutant 1 GATTCGCTGAtcTCCATGAGAAAGCTT + Site IV mutant 2 GATTCGCTcAgcaCCATGAGAAAGCTT + Site IV mutant 3 GATTCGCTGACGTCagaGAGAAAGCTT YY1 consensus CGCTCCGCGGCCATCTTGGCGGCTGGT ++++ YY1 mutant CGCTCCGCGattATCTTGGCGGCTGGT Solid lines above and below sequences indicate, respectively, the ATF and YY1 consensus motifs (32, 41, 42). Column 3 provides an assessment of the effects of mutations on the binding of NMP-1, with relative strength of binding designated as follows: -, no binding; ±, severely reduced binding; + + + or + + + +, strong binding. Downloaded by guest on September 29, 2021 10528 Cell Biology: Guo et aL Proc. Natl. Acad. Sci. USA 92 (1995)

A Site - ATF AP-1 To establish directly that NMP-1 is identical to YY1, we analyzed binding of purified YY1 protein to site IV. The gel-shift pattern (Fig. 4) shows that YY1 binds to both the YY1 consensus sequence and site IV. More importantly, when analyzed with the same probe (site IV), the gel-shift complexes formed by NMP-1 isolated from the HeLa nuclear matrix and by purified, bacterially expressed YY1 exhibit equivalent elec- trophoretic mobilities. Thus, purified YY1 protein reconsti- NMP-1-- tutes the NMP-1-site IV complex. As an additional confirma- tion of the identity of NMP-1 and YY1, we probed size- fractionated, membrane-immobilized nuclear matrix proteins aihI~~~~~~~~~~miuwImM~~~~~~~~~~~~~~~~iu~~~ with radiolabeled YY1 and site IV oligonucleotides (South- western blotting). As shown in Fig. 5, both oligonucleotides interact specifically with a protein of 68 kDa, the molecular mass of YY1. Our combined results from oligonucleotide competition, antibody reactivity, reconstitution with purified mass analysis of DNA binding B M Site IV c mut 1 E mut 2 i mut3 protein, and molecular activity establish that NMP-1 is identical to YY1.

C TGACGTCCATGC) TGAT-CTCCATGAC TC TC-AGCACCATGC/1 TGACGTCAGAG Partitioning of NMP-l/YY1 Between Nuclear Matrix and Nonmatrix Nuclear Compartments. The result that NMP-1 is identical to YY1 raises several questions. For example, YY1 has previously been isolated from intact nuclei by extraction al. q with 0.42 M KCI according to the method of Dignam et (38). NMP-1 -_ ad NW _- ms N mu Wm am This nuclear extraction procedure yields many chromatin- associated ubiquitous transcription factors, including YY1, by dissociation with high salt. In contrast, NMP-1/YY1 is isolated from nuclear matrices after removal of chromatin by extensive DNase I digestion and 0.25 M ammonium sulfate extraction; FIG. 2. Competition of NMP-1 binding activity with different NMP-1/YY1 is released only when the nuclear matrix is transcription factor binding sites and site IV mutants. HeLa nuclear solubilized with 8 M urea. Based on these different charac- matrix proteins (1 gg) were incubated with 20 fmol of site IV probe. Competing oligonucleotides were added at 50-, 100-, and 250-fold teristics, the possibility arises that YY1 consists of subpopu- molar excess. No competitor was added to the control (CTRL). A and lations with different biochemical properties and/or partitions B show competition experiments with wild-type transcription factor in distinct nuclear compartments. binding sequences and site IV mutants, respectively (see Table 1). Gel-shift assays performed in the presence of the YY1 consensus oligonucleotide or YY1 antibody (Fig. 3) indicate binds to the wild-type but not to a mutant YY1 binding site. that the NMP-1/YY1 protein from nuclear matrix prepara- The gel-shift immunoassays show that when nuclear matrix tions is indistinguishable from YY1 present in nuclear extracts. proteins were incubated with a polyclonal antibody directed These results are consistent with previous studies in which against the DNA binding domain of YY1, the NMP-1-related binding activities were observed in both prep- NMP-1-site IV complex was disrupted. The antibody inhibited arations (31). Western blot results (Fig. 5A) show that a 68-kDa NMP-1 binding to the oligonucleotide, consistent with its protein that is immunoreactive with the same YY1 antibody recognition of an epitope within the DNA binding domain; used in the supershift experiments is present in both nuclear limited formation of a ternary DNA-protein-antibody com- matrix and nonmatrix nuclear preparations. Southwestern plex (supershift) was also detected. No immunoreactivity for blotting results confirm that the 68-kDa protein exhibits NMP-1 was observed with an extensive panel of antibodies specific recognition for both site IV and YY1 consensus directed against the ATF and AP1 family of proteins (data not oligonucleotides (Fig. 5B). shown). In summary, the results obtained with oligonucleotide NMP-1/YY1 Is Present in the Nuclear Matrix. To address competition and gel-shift immunoassays indicate that NMP-1 where YY1 is localized in situ, we performed a series of contains YY1-related DNA binding activity and is immuno- immunofluorescence experiments with a panel of antibodies logically related to YY1. directed against YY1, Numatrin/B23, lamin B, and NuMa. B ROS 17/2.8 A HeLa NM NE I _1- .n w NM NE - = m n: o O: o O >- o e >- E -j -j e C > FIG. 3. Identification of NMP-1 as tran- > o o o oo> H >- >- L C F > >- C scription factor YYl. Nuclear matrix (NM) (1 o >- >- < < L)> > < < < j,ug) as well as nuclear extract (NE) (1 jig) < < <; r<< 2 * proteins from HeLa (A) or ROS 17/2.8 (B) cells were incubated with site IV probe in the C. absence or presence of a 25-fold molar excess of ATF, AP1, or YY1 consensus oligonucle- C ?0 ATE-1 E -' otides or the corresponding YY1 mutant oli- ATF-1 E gonucleotide (mutated at the CCAT core NMP-1 - LiI motif). ATF-1-related complexes (bracket) NMP-1- 0 idw ii are selectively blocked upon ATF-1 antibody addition (data not shown). The NMP-1 com- i. plex (NMP-1) and an ATF-related complex s i i s s (C) are also indicated. A limited amount of L YY1 ternary antibody-protein-DNA com- a"' plex (supershift) is formed (S). Downloaded by guest on September 29, 2021 Cell Biology: Guo et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10529

32 P-YYl cons 32lSITE IV

- YY1 >- YY1 >- i YY1 >- .. F cons .C F- Fc cons o- 0 c FIG. 6. YY1 is present in nu- His-YY1 + + + + *,- , 8'a*';}...... *'.'. + ++ + - cleolar regions of the nuclear ma-

NM ------+ + + + trix. CaSki cells were treated in situ to remove soluble compo- nents and chromatin. After fixa- tion, cells were stained with a rabbit polyclonal antibody against I. -*Super- YY1 and a mouse monoclonal an- shift tibody against Numatrin/B23 and fi..g *" with species-specific second anti- * NMP1 bodies labeled with rhodamine and fluorescein, respectively. A phase-contrast image of the stained cell is shown inA. YY1 (B) was present primarily in the rem- nant nucleoli, where its distribu- tion overlapped that of Numatrin/ B23 (C). Additional smaller do- mains containing YY1 can be observed. Some of these also con- tain B23 and may be smaller nu- FIG. 4. Purified YY1 reconstitutes the NMP-1 complex with site cleoli. Some of the smaller YY1- IV. Bacterially expressed and purified YY1 protein (1 ,g) was containing domains do not have incubated with the site IV or YY1 consensus probe in the absence or detectable Numatrin/B23 and re- presence of the unlabeled YY1 consensus oligonucleotide as compet- main uncharacterized. itor. In addition, binding reactions were performed with site IV probe and HeLa nuclear matrix (NM) proteins. Immunoreactivity of the which was more uniformly distributed. Additional small and NMP-1 and YY1 complexes was also assessed. discrete YY1-containing domains were observed outside the larger remnant nucleoli. Some of these also contained B23 and Studies of detergent-permeabilized HeLa cells showed that the could represent small nucleoli, while others had no observable localization of YY1 was mostly nucleolar and was distinct from Numatrin/B23. Thus, YY1 is an integral component of the that of lamin B and NuMa (data not shown). Lamin B antibody internal nuclear matrix protein network. resulted in relatively uniform nuclear perimeter staining, Inouye and Seto (45) have shown that YY1 can interact consistent with localization of lamin B within the lamina-pore functionally with the nuclear matrix protein Numatrin/B23; complex (43), whereas immunostaining with the NuMa anti- B23 is capable of releasing the repressive effect ofYY1 on gene body was uniformly distributed throughout the nucleoplasm transcription. The subcellular location where these interac- but was not observed in nucleoli (44). tions occur remains to be established. Our immunofluores- We then examined nuclear matrix-intermediate filament cence results show that Numatrin/B23 is concentrated in the preparations in HeLa and CaSki cells in situ, after removal of nucleolus (Fig. 6C) both in detergent-permeabilized cells and cytoplasm and the majority of chromatin. Our experimental in the nuclear matrix. This result suggests that the nuclear results clearly show the presence of YY1 in the nuclear matrix, matrix-associated residual nucleolus represents one potential primarily associated with nucleolar regions (Fig. 6A and B), as location for interactions between YY1 and B23. can be seen by its colocalization with the nucleolar protein Numatrin/B23 (Fig. 6C). YY1 was not uniformly distributed in DISCUSSION the nucleolus but was concentrated in smaller subnucleolar domains. These domains were not seen for Numatrin/B23, In this study we have shown that the transcriptional regulator YY1 is an intrinsic component of the nuclear matrix. This A B finding corroborates recent evidence suggesting that a specific 2 wI subset of transcription factors are sequestered in the nuclear kD Z Z kD 1 2 3 matrix (23-25, 46, 47) and that this localization may be 4- functionally linked to regulation of gene expression (3). We 84- 8 _ have previously proposed that the presence of transcription fAmo*o& -*--YY1 /NM P 1 53- t -YY1/NMP1 factors in the nuclear matrix may facilitate transient gene- 53- 3 5- matrix interactions (3, 23, 24). This type of gene association 35- with the nuclear matrix is different from the stable attachment of genes to the nuclear matrix via MARs, which span large FIG. 5. Transcription factor YY1 is present in the nuclear matrix A+T-rich DNA sequences (between 0.1 and 1.0 kb) and (NM) and nonmatrix (NE) nuclear fractions. (A) Western blot analysis topoisomerase recognition sites (9-18). Whereas MARs are was performed with nuclear matrix (10 ,ug) and nonmatrix nuclear (30 thought to confer position-independent gene transcription by jLg) protein from HeLa cells. Detection of YY1 was achieved by using generating functional chromatin-loop domains, nuclear ma- an antibody directed against the C terminus of YY1 (68 kDa). Two trix-associated transcription factors may affect gene regulation bands of lower molecular mass may represent truncated C-terminal modulating chromatin topology via transient teth- fragments of YY1. As the C terminus of YY1 contains the DNA by locally binding domains, similar bands are also detected by Southwestern ering of DNA to the matrix (3, 4, 48). blotting analysis (see below). (B) Southwestern blot analysis was The presence of YY1 in both the nuclear matrix and performed with the YY1 consensus (lane 1) or site IV (lane 2) nonmatrix nuclear fractions is consistent with the subnuclear oligonucleotide used as probe. The ATF consensus probe (lane 3) distribution of several ubiquitous transcription factors (23-25), served as a control for nonspecific binding. as well as c-myc (49), ElA (50), the retinoblastoma gene Downloaded by guest on September 29, 2021 10530 Cell Biology: Guo et al. Proc. Natl. Acad. Sci. USA 92 (1995)

product Rb (51), and Numatrin/B23 (52). These regulatory 16. Dickinson, L. A., Joh, T., Kohwi, T. & Kohwi-Shigematsu, T. factors, including YY1, may be subject to dynamic shuttling (1992) Cell 70, 631-645. between distinct subcellular compartments, perhaps to accom- 17. Chou, R. H., Churchill, J. R., Flubacher, M. M., Mapstone, D. E. modate location-dependent regulatory functions. For exam- & Jones, J. (1990) Cancer Res. 50, 3199-3206. 18. Patriotis, C., Andreeva, M., Pascaleva, M., Ivanov, V. & Djond- ple, the Gl-related, hypophosphorylated form of Rb is present jurov, L. (1990) J. Cell Sci. 95, 667-674. in the nuclear matrix, but, after phosphorylation during late G1, 19. Xing, Y., Johnson, C. V., Dobner, P. & Lawrence, J. (1993) Rb is no longer associated with the nuclear matrix (51). Modifi- Science 259, 1326-1330. cations in the representation of fos/jun proteins in the nuclear 20. Huang, S. & Spector, D. L. (1992) Proc. Natl. Acad. Sci. USA 89, matrix and nonmatrix nuclear fractions in osteoblasts are related 305-308. to phenotypic properties (23). These observations support the 21. Nickerson, J. A., Krochmalnic, G., Wan, K. M. & Penman, S. concept that posttranslational modification may affect subnuclear (1989) Proc. Natl. Acad. Sci. USA 86, 177-181. partitioning as well as the function of regulatory molecules. 22. Harris, S. G. & Smith, H. C. (1988) Biochem. Biophys. Res. Association of transcription factors with the nuclear matrix Commun. 152, 1383-1387. may involve specific acceptor proteins. For example, the 23. van Wijnen, A. J., Bidwell, J. P., Fey, E. G., Penman, S., Lian, J. B., Stein, J. L. & Stein, G. S. (1993) Biochemistry 32, 8397-8402. nuclear matrix structural protein lamin A/C has been shown 24. Bidwell, J. P., van Wijnen, A. J., Fey, E. G., Dworetzky, S., to interact with Rb, providing a potential mechanism for Penman, S., Stein, J. L., Lian, J. B. & Stein, G. S. (1993) Proc. tethering Rb to the nuclear matrix (51). Evidence for an Natl. Acad. Sci. USA 90, 3162-3166. acceptor protein mediating YY1 association with nuclear 25. Sun, J. M., Chen, H. Y. & Davie, J. R. (1994)1. Cell. Biochem. 55, matrix remains to be established. However, YY1 is capable of 252-263. interacting with Numatrin/B23 (45), c-myc (35), and ElA (34); 26. Alexander, R. B., Greene, G. L. & Barrack, E. R. (1987) Endo- each of these proteins has been shown to associate with the crinology 120, 1851-1857. nuclear matrix. Furthermore, the ubiquitous transcription 27. Schuchard, M., Subramaniam, M., Ruesink, T. & Spelsberg, T. C. factors Sp-1 and TFIIB are capable of interacting with YY1 (1991) Biochemistry 30, 9516-9522. 28. van Steensel, B., Jenster, G., Damm, K., Brinkmann, A. 0. & van (36, 53). Taken together, these observations suggest that YY1 Driel, R. (1995) J. Cell. Biochem. 57, 465-478. may mediate gene-matrix association by direct interactions 29. Bidwell, J., van Wijnen, A. J., Fey, E. G., Merriman, H., Penman, with YY1 consensus elements in gene promoters and indirectly S., Lian, J. B., Stein, J. L. & Stein, G. S. (1994) J. Cell. Biochem. by protein-protein interactions with other DNA binding tran- 54, 494-500. scription factors that are nuclear matrix associated. 30. Pauli, U., Chiu, J. F., Ditullio, P., Kroeger, P., Shalhoub, V., Interestingly, we have also observed that both YY1 and Rowe, T., Stein, G. & Stein, J. (1989) J. Cell. Physiol. 139, Numatrin/B23 are localized and concentrated in the nucleo- 320-328. lus. Numatrin/B23 is a phosphoprotein involved in rRNA 31. Dworetzky, S. I., Wright, K. L., Fey, E. G., Penman, S., Lian, transport between the nucleus and the cytoplasm (54). YY1 J. B., Stein, J. L. & Stein, G. S. (1992) Proc. Natl. Acad. Sci. USA 89, 4178-4182. may therefore be involved in the regulation of RNA poly- 32. Shrivistava, A. & Calame, K. 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