Identification of a Candidate Regulatory Region in the Human CD8 Complex by Colocalization of DNase I Hypersensitive Sites and Matrix Attachment Regions Which Bind This information is current as SATB1 and GATA-3 of September 28, 2021. Lynda J. Kieffer, John M. Greally, Inna Landres, Shanta Nag, Yuko Nakajima, Terumi Kohwi-Shigematsu and Paula B. Kavathas

J Immunol 2002; 168:3915-3922; ; Downloaded from doi: 10.4049/jimmunol.168.8.3915 http://www.jimmunol.org/content/168/8/3915 http://www.jimmunol.org/ References This article cites 65 articles, 35 of which you can access for free at: http://www.jimmunol.org/content/168/8/3915.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Identification of a Candidate Regulatory Region in the Human CD8 Gene Complex by Colocalization of DNase I Hypersensitive Sites and Matrix Attachment Regions Which Bind SATB1 and GATA-31

Lynda J. Kieffer,* John M. Greally,‡ Inna Landres,* Shanta Nag,* Yuko Nakajima,¤ Terumi Kohwi-Shigematsu,¤ and Paula B. Kavathas2*†

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs

facilitate long-range remodeling required for enhancer activity and have been found closely linked to several lymphoid Downloaded from enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3؅ end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding , SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs http://www.jimmunol.org/ capable of binding SATB1 and GATA-3 at the 3؅ end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression. The Journal of Immunology, 2002, 168: 3915Ð3922.

s thymocytes progress through development, they un- suggest coordinate regulation. However, regulation is not always dergo induction and repression of a number of cell sur- coordinated, particularly in cells which seem to be extrathymically face molecules. Changes in the expression of the CD4 derived, as subsets of human NK cells (4) and gut intraepithethelial A 3 and CD8 T cell surface glycoproteins best characterize the stages lymphocytes (IELs) (5, 6) express only the CD8␣␣ homodimer. of ontogeny and ultimately define the two major lineages of mature Toward identifying cis-acting transcriptional regulatory ele- T cells (CD4ϩCD8Ϫ or CD4ϪCD8ϩ). Gene knockout studies with ments in the CD8 loci, large fragments of genomic DNA have been by guest on September 28, 2021 CD4 and CD8␣ have shown that the entire respective lineage does used to make transgenic mice. A 95-kb human genomic fragment not develop (1, 2), making it likely that factors that regulate CD4 beginning ϳ25 kb upstream of the CD8B gene and containing the and CD8 expression also regulate the functional commitment of entire CD8B gene afforded developmentally correct expression on the cell. Therefore investigating the mechanisms controlling CD4 thymus-derived T cells in transgenic mice, indicating that CD8 and CD8 expression should increase our understanding of thymo- lineage-specific regulatory sequences must be located within that cyte development. fragment (7). Likewise, an 80-kb murine genomic fragment from CD8 can be expressed in mice and humans as an ␣␣ homodimer 2 kb upstream of the CD8B gene to 25 kb downstream of the or an ␣␤ heterodimer. Human CD8␤ can also be expressed as a ␤␤ CD8A gene allowed appropriate expression in transgenic mice (8). homodimer in transfected COS cells and transgenic mice (3). The The 80-kb murine genomic fragment contained four clusters of CD8A and B , closely linked at a distance of ϳ36 kb in mice DNase hypersensitive (HS) sites (9) which were analyzed for en- and 56 kb in humans (see Fig. 1), are coexpressed on most CD8ϩ hancer activity in transgenic mice. One cluster at the 3Ј end of the T cells. The close linkage and coexpression of the CD8 genes CD8B gene and two in the intergenic region had enhancer activity. The results indicate that there are separate elements for CD8 ex- pression in the thymus vs the periphery, and possibly also for *Department of Laboratory Medicine and †Department of Genetics and Section of CD8␣ vs CD8␤ (9Ð13). ‡ Immunobiology, Yale University, New Haven, CT 06520; Department of Medicine, To locate regulatory regions within the human CD8 gene com- Division of Hematology, Albert Einstein College of Medicine, Bronx, NY 10461; and ¤Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720 plex, we mapped the DNase HS sites which often colocalize with Received for publication December 10, 2001. Accepted for publication February cis-acting transcriptional regulators. In addition, since many lym- 20, 2002. phoid gene enhancers are closely linked to matrix association re- The costs of publication of this article were defrayed in part by the payment of page gions (MARs) (14), we also undertook the mapping of MARs in charges. This article must therefore be hereby marked advertisement in accordance the human CD8A and B loci. MARs, interspersed in genomes on with 18 U.S.C. Section 1734 solely to indicate this fact. the average of 50Ð100 kb, have been identified as specialized 1 This work was supported by National Institutes of Health Grant CA48115 (to P.B.K.), National Research Service Award 5 F32 AI09700 (to L.J.K.), National In- genomic sequences that tightly associate with the nuclear matrix, stitutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases a RNA and protein containing fraction that remains after high salt Grants DK02467 and DK45676 (to J.M.G.), and National Institutes of Health Grant R01 CA39681 (to T.K.-S.). 2 Address correspondence and reprint requests to Dr. Paula B. Kavathas, 333 Cedar Street, P.O. Box 208035, New Haven, CT 06520-8035. E-mail address: paula. 3 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; MAR, matrix [email protected] attachment region; HS, hypersensitive; LFE, low energy free; GC, guanine cytosine.

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 3916 HUMAN CD8 CANDIDATE REGULATORY REGION BINDS SATB1 and GATA-3 extraction and DNase I treatment. MARs serve to anchor chroma- nuclear matrices at room temperature with 100 or 200 ␮g of unlabeled tin loops to the nuclear matrix and have been shown to facilitate Escherichia coli DNA as a nonspecific competitor, labeled plasmid frag- long-range chromatin remodeling and accessibility (15, 16). We ments were added and the incubation was continued for 2 h. Insoluble matrix were pelleted and washed to remove unbound DNA, treated identified several MARs spanning the human CD8 loci. Interest- with proteinase K, and the remaining DNA was electrophoresed on agarose ingly, three strong MARs are closely linked to DNase HS clusters gels. Gels were dried on Whatman 3 MM paper (Whatman, Clifton, NJ) III and IV, located at the 3Ј end of the CD8B gene, making this and exposed to X-OMAT AR film. A 6-kb HindIII fragment from the p34 region a prime candidate for an element regulating CD8 gene region upstream of ZNF 127, which contains strong MARs on 2.7- and 3.3-kb fragments generated by BamHI digestion (35, 36), was used as a expression. positive control in these assays. The vector DNA from which the test frag- To further establish the significance of this region, we analyzed ments were excised served as an internal negative control. To show the the candidate regulatory region for binding of SATB1, a tissue- relative intensities and sizes of the input radiolabeled probe fragments, 5% specific MAR-binding protein (17). Studies of SATB1 knockout of the total radioactivity added to each MAR-binding assay (the probe) was animals showed that SATB1, expressed primarily in thymocytes, electrophoresed beside 50% of the bound fragments. The intensities of the insert (Ii) and plasmid bands (Ip) within each assay were quantified by regulates temporal and spatial expression of multiple genes during ϩ densitometry using the NIH Image program (rsb.info.nih.gov/nih-image). T cell development, and is required for maturation of CD4 and Adjusted relative binding ratios were calculated according to the following ϩ ϫ CD8 T cells (18). Within the MARs associated with the human equation (37): (Iibound/Ipbound) (Ipprobe/Iiprobe). CD8 DNase HS clusters III and IV, we found two fragments that Sequence analysis bound SATB1 with high affinity in an EMSA. Further analysis of the sequence in the region of HS clusters III Base composition (Percent A ϩ T) plots were produced using the MacVec- and IV revealed several potential binding sites for GATA-3, a tor software program (Accelrys, Princeton, NJ) with a window size of 50 Downloaded from nt. Maps of MAR-binding motifs were created by entering the specific widely expressed in embryonic tissues, but motifs (38) into an enzyme filter in the MapDraw program (version 4.0) of whose expression is mainly limited in adult animals to T cells and Lasergene software (DNAstar, Madison, WI). Repetitive content in each se- NK cells (19Ð21). Targeted deletion of GATA-3 was embryoni- quence was identified using the RepeatMasker database (http://ftp.genome. cally lethal (22), but using Rag complementation, it was deter- washington.edu/cgi-bin/RepeatMasker/). Free energy values were calculated with the nearest-neighbor algorithm program Web-Thermodyn (http://wings. mined that GATA-3 is required for development of the earliest T buffalo.edu/gsa/dna/dk/WEBTHERMODYN/index.html) using the program’s cell progenitors (23). GATA-3 binding sites have been reported in default values. We have previously found that the presence of free energy http://www.jimmunol.org/ several T cell-specific genes (24Ð31), including the murine CD8 values of Ͻ90 in four or more successive windows was a specific indicator of promoter/enhancer (32). In the present study, EMSA analysis re- in vitro MARs at the ␣ and ␤ globin and imprinted loci (J. M. Greally, un- vealed that an oligonucleotide with a double GATA-3 binding site, published data). The several sequences fitting this criterion are indicated as low free energy (LFE) regions in Fig. 4. corresponding to the cluster IV HS site located in a strong MAR, binds GATA-3 strongly, while others in the HS cluster III and IV EMSAs region also bind. Colocalization of GATA-3 binding sites, SATB1 The SATB1 gel shift assay was performed as described previously (39), binding sites, MARs, and DNase HS clusters suggests this region using recombinant SATB1, a GST fusion protein consisting of aa 346Ð763 is a candidate regulator of human CD8 expression. of human SATB1 (GST-SATB1). This protein contains both the MAR- by guest on September 28, 2021 binding domain and homeodomain. To estimate the Kd values for SATB1- Materials and Methods binding sites, gel shift assays were run using 0.5 ng of DNA probe and increasing concentrations of SATB1. The dried gels were exposed to a Cell lines phosphor imager screen, and the amounts of remaining free probe in each All cell lines were grown in RPMI 1640 medium supplemented with 2 mM lane were quantitated. The Kd values were determined by calculating the L-glutamine, 100 U of penicillin/ml, 100 ␮g of streptomycin/ml, and 10% concentration of SATB1 required to shift 50% of the probe DNA. FCS. Cell lines used included the EBV-transformed B cell line UC and the The GATA-3 gel shift assays were performed essentially as described human T cell lines HPB.ALL (CD4ϩCD8␣ϩ␤ϩ), JM (CD4ϩCD8␣-␤Ϫ), previously (27). The binding reactions contained 10 mM HEPES (pH 7.9), ϩ Ϫ and Jurkat (CD4 CD8 ). Cells were cultured at 37¡C in a water-saturated 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mM MgCl2, 10% glycerol, 50 ␮ ␮ ϫ atmosphere of 5% CO . g/ml poly(dI)-poly(dC), 200 g/ml BSA, 1 protease inhibitor mixture 2 (catalogue no. 1836170; Roche, Mannheim, Germany), and nuclear extract DNase I hypersensitivity mapping prepared from Jurkat cells stimulated for 8 h with 1 mM Bt2 cAMP and 25 ng/ml PMA. Abs used for the supershifts were anti-GATA-3 (Santa Cruz DNase I hypersensitivity mapping was performed as described previously Biotechnology, Santa Cruz, CA) and an isotype control, anti-human CD8␤ (7). Briefly, DNA purified from DNase-treated nuclei was restriction di- (Immunotech, Westbrook, ME). The top strand sequences of the oligonu- gested and used to prepare Southern blots which were probed with the cleotides used in the GATA-3 EMSAs are shown below. The number in following fragments (see Fig. 1): 1, 0.9-kb NotI-EcoRI fragment from the parentheses indicates the location in plasmid 1231 for oligonucleotides 1Ð3 Ј Ј 3 end of clone 647; 2, 0.5-kb NcoI-BamHI fragment from the 3 end of and in plasmid 1230 for oligonucleotides 4Ð7. Bold sequences highlight Ј clone 646; 3, a 0.8-kb BamHI-KpnI fragment from the 5 end of clone the putative GATA-3 motif, and underlined sequences indicate that the 1229; 4, 0.9-kb KpnI-Stu fragment from 1229; 5, 1-kb EcoRV-BamHI frag- motif is on the complimentary strand: 1, atctccatcagatctcttgggtg (3858); Ј ment from the 3 end of clone 1231; 6, 0.6-kb Xho-BamHI fragment from 2a, ctctctccatatcagcaataag (541); 2b, tcgttttcttatcattcatgtg (4569); 3, tgcat Ј ␣ the 3 end of 1230; and 7, 0.6-kb BssHII fragment including CD8 exon II. accctatcttgaaatttgtgggt(5844); 4, ttgaaaagagatctaaaattga (2909;, 5, caaaaa Fragments 1, 2, 3, 5, and 6 were used to probe DNA restriction digested ttagatcaagtagataaaattta (5013); 6, aaaatttgttatcaccttttaa (5262); and 7, with BamHI, fragment 4 with Kpn-digested DNA, and fragment 7 with tattatgcagatatcagataagattcatgaag (5400) contains three GATA-3 motifs. BglII-digested DNA. Fragment 6 was also used to probe DNA double The sequence of the IL-5 promoter GATA-3-positive control oligonucle- digested with Xho and SphI. otide is cctctatctgattgttagca (27). MAR assays The GenBank accession number for the 19-kb nucleotide sequence from the 3Ј end of the human CD8B gene is AY032722. Nuclei from the human T cell line JM (CD4ϩCD8␣ϩ␤Ϫ) were obtained by hypotonic lysis and purified by centrifugation through a cushion of 2 M Results sucrose. Nuclear matrices were isolated in the continuous presence of 250 DNase hypersensitivity mapping ␮M PMSF and 10 ␮g/ml leupeptin, as described previously (33, 34). Briefly, isolated nuclei were digested with 100 ␮g/ml DNase I in 10 mM We had previously identified a tissue-specific DNase HS cluster NaCl, 3 mM MgCl2, 10 mM Tris (pH 7.4), 0.25 mM sucrose, and 1 mM upstream of the human CD8B gene (HS I) (7) and another in the CaCl2 for1hatroom temperature, and nonmatrix proteins were extracted with 2 M NaCl. Nuclear matrix binding was determined in an in vitro last intron of the CD8A gene (HS VI) (40). The entire human CD8 DNA-binding assay (34). Briefly, plasmids were restriction digested and gene complex was subcloned to facilitate a comprehensive map- the fragments were end-labeled with [␥-32P]ATP. After preincubation of ping of DNase HS sites (Fig. 1). We determined that these clones The Journal of Immunology 3917

FIGURE 1. Map of the human CD8A and CD8B gene complex (bottom) with a partial restriction map of the enzymes used to map DNase HS sites (B, BamHI; K, Kpn;X,Xho; Bg, BglII; S, Sph). Subclones used in this work are shown. DNase HS mapping was performed on the 95-kb region flanked by and included within clones 647 and 732. The only regions not covered in this DNase I hypersensitivity mapping survey are the 355-bp PCR product generated from the region between clones 1231 and 1230 and a 2-kb fragment on the 3Ј end of plasmid 647. Locations of the DNase HS sites are shown,

the tissue-specific sites with an arrow. Probes used to identify HS sites are indicated. MAR mapping was performed on the 102-kb region flanked by and Downloaded from included within clones 1235-4 and 732 with the exception of the 355-bp PCR product, which was not AT rich. The locations of the MARs are identified, with strong MARs shown as f, weak MARs with u. contain the whole CD8 gene complex, except for a small region not seen in UC cells, whereas sites at 3.2 and 3.5 kb from the 3Ј between clones 1231 and 1230 containing CD8␤ exon IX, which end of 646 were also seen in UC cells. Cluster II contains addi- we obtained by PCR. Cell lines used in this work were HPB.ALL, tional sites, which map to 3.5, 3.0, 1.7, 1.6, 1.3, and 1.1 kb from http://www.jimmunol.org/ which are a CD4ϩCD8␣ϩCD8␤ϩ thymoma cell line, and UC the 5Ј end of 1229. Of these, only the 3.5-kb band appeared to be cells, a B cell line, as a control for tissue specificity. In the present specific for HPB.ALL cells. work, we identified four additional DNase HS clusters in the hu- In summary, we identified four new DNase HS clusters in the man CD8 loci, making a total of six HS clusters (Fig. 1). human CD8 gene complex. These are DNase HS cluster II (HS II), The data for DNase HS cluster III, located between CD8␤ exons located just downstream of CD8␤ exon VII, HS III, between VIII and IX, are shown in Fig. 2A. Of the seven bands marked, CD8␤ exons VIII and IX, and HS IV and V, in the intergenic those at 3.7 and 3.4 kb appeared to be specific for HPB.ALL cells. region, 3Ð5 and 8Ð11 kb downstream of CD8␤ exon IX, respec-

The 5.6- and 4.5-kb bands can be seen in the UC cells in Fig. 2A, tively. We also observed four weak but tissue-specific DNase HS by guest on September 28, 2021 and bands appearing to correspond to the 3-, 2.4-, and 1.3-kb bands sites 0Ð0.8 kb upstream of the first exons of both CD8A and were seen in other blots made from UC cells treated with 25 U/ml CD8B, in the promoter regions (Fig. 1 and data not shown). DNase (data not shown). The data for DNase HS cluster IV and part of cluster V are MAR assays shown in Fig. 2B. Of the bands seen, those at 4.7, 3.8, and 2.9 kb An in vitro MAR-binding assay was used to map the MARs in the appeared to be specific for HPB.ALL cells, since those at 0.7, 0.5, CD8 loci. Over the whole region, there were four strong MARs and 0.4 were also seen in UC cells. These latter three small bands and several weaker MARs. In plasmid 1235-4, located 13Ð17 kb at the 3Ј end of 1230 have been placed in cluster V in Fig. 1 upstream of the CD8B gene, there is a strong MAR in the 5Ј 2-kb because they are closer to the other sites in cluster V than they are fragment and, in addition, at least one weaker MAR (Fig. 3A). to the three tissue-specific DNase HS sites in cluster IV. There are Since the 0.9- and 1.6-kb fragments are juxtaposed, it is possible several more weak DNase HS sites within cluster V, seen at 1.3, that there is only one MAR split between them. Another strong 1.0, 0.8, 0.5, 0.3, 0.25, and 0.1 kb from the 5Ј end of 1264 (data not MAR, located between CD8B exons VIII and IX in fragment shown). These bands, although weak, appear to be tissue specific. 1231, is shown in Fig. 3B. This strong MAR is closely linked to the There are several DNase HS sites within cluster II (Fig. 1 and tissue-specific DNase HS sites in cluster III (Fig. 4A). There is also data not shown). Sites which map to 1.5, 1.1, and 1.0 kb from the a weaker MAR at the 5Ј end of clone 1231. The data for clone 3Ј end of clone 646 appear to be tissue specific, in that they were 1230, shown in Fig. 3C, indicate a cluster of MARs in the center

FIGURE 2. DNase I hypersensitivity mapping of the re- gion contained in clone 1231 (A) and clone 1230 (B). Nuclei from the human cell lines HPB.All or UC were either un- treated or treated with increasing concentrations of DNase I before isolation of genomic DNA. DNase concentrations in units per milliliter are indicated at the top of each lane. The isolated DNA was cut with the restriction endonuclease BamHI and Southern blot analysis performed using a 32P- labeled probe isolated from the 3Ј end of clone 1231 (A)or 1230 (B) (see Fig. 1). The locations of the HS sites are in- dicated to the right of each gel, and represent distance in kilobases from the probe. Bands marked with arrows are those which appear to be tissue specific. 3918 HUMAN CD8 CANDIDATE REGULATORY REGION BINDS SATB1 and GATA-3

of plasmid 1230, with two strong MARs flanking a weaker one. Two of the three tissue-specific DNase HS sites of cluster IV are located within the weak MAR and the third is within the strong MAR on the 3Ј end of 1230 (Fig. 4B). We found six additional weaker MARs in the CD8 complex (data not shown). Thus, dis- tributed over the CD8 gene complex were 4 strong MARs and 10 weaker MARs, as indicated in Fig. 1.

Sequence analysis Given that MARs are generally AT-rich sequences, we considered whether this frequency of MARs could be attributed to the human CD8 genes possibly being located in an AT-rich isochore, as was the case for the 15q11-13 imprinting center (35). We determined the isochore type to which the CD8 genes belonged by manually scanning the third base of each codon in the coding regions for guanine cytosine (GC) content (percent GC3). CD8A and B genes were grouped into their corresponding isochores based on the fol- lowing criteria: L1 ϩ L2, GC poor (GC3 Ͻ 57%), H1 ϩ H2, GC high (57% Ͻ GC3 Ͻ 75%), and H3, GC rich (GC3 Ͼ 75%) (41, Downloaded from 42). The GC3 content of the CD8A and B genes were 71 and 84%, placing them well into the GC high and GC-rich isochores, re- spectively (data not shown). This indicates that the frequency of MARs we observe is not simply due to location of the CD8 genes in an AT-rich isochore.

Since clones 1231 and 1230 contain strong MARs closely linked http://www.jimmunol.org/ to tissue-specific DNase HS sites, we considered them likely can- didates for containing regulatory elements. Therefore, we se- quenced the 19 kb spanned by the plasmid 1231, plasmid 1230, the PCR product between 1231 and 1230, and 4 kb from the 5Ј end of 1264. We located the CD8␤ exons VIII and IX within this region as shown in Fig. 1. The sequence of a typical MAR is ϳ65% AT rich (43) and contains a region of 150Ð200 bp that has a high potential for base by guest on September 28, 2021 unpairing (44Ð46). A Thermodyn analysis of the 19-kb sequence predicted seven potential LFE regions (Fig. 4). Only one of the seven LFE regions did not localize to the biochemically determined MARs. A mapping of repetitive elements in clones 1231 and 1230 is shown in Fig. 4. There were eight whole or partial Alu elements. Interestingly, five of the six long interspersed nuclear elements were within fragments that bound to the nuclear matrix. The strong MAR in 1231 overlapped on its 5Ј end with a Tigger 1 element, an interspersed repeat that resembles DNA transposons, which move by excision and reintegration into the genome without a RNA intermediate. Several sequence motifs have been associated with MARs (17, 37, 44, 47Ð49). We mapped ATATTT and vertebrate topoisom- erase sites and found that there was some clustering in the bio- chemically defined MARs, but this clustering was not absolute, particularly for the topoisomerase (Fig. 4, A and B). In addition, we analyzed the sequence for potential SATB1-binding sequences. SATB1 is an interesting MAR-binding protein in that it does not recognize a DNA sequence but rather it is believed to recognize ATC sequences indirectly by the altered sugar-phosphate back- bone determined by the ATC sequence context (50). The ATC

FIGURE 3. Nuclear matrix binding assays (MAR assays) using as probes restriction-digested plasmids 1235-4 (A), 1231 and 1313 (B), and enzymes, that were tested for MAR activity. The relative binding ratios (RBR) 1230 and 508 (C). Locations of these plasmids within the CD8 complex are of the fragments within each assay are indicated beneath the restriction maps. shown in Fig. 1. The three lanes in each assay represent, from left to right, Quantitation of some bands was not possible if they were not well resolved unbound end-labeled probe, and the fraction that bound to the probe in the from other bands on the gel. Strong and weaker MARs are indicated in presence of 100 or 200 ␮g/ml competitor E. coli DNA. Band sizes are these maps by dark and light shading, respectively. B and C, A compilation indicated to the right of each gel. P, plasmid band. Below the gels are of the locations of the MARs is shown at the bottom. C, An additional restriction maps showing the fragments, generated by each set of restriction analysis using BamHI/BglI-digested plasmid 1230 as probe is not shown. The Journal of Immunology 3919 Downloaded from FIGURE 4. Sequence analysis of clones 1231 (A) and 1230 (B). The top of this figure shows the locations of the MARs as determined in Fig. 3. Dark and light shading indicate strong and weaker MARs, respectively. Locations of DNase HS sites, as determined in Fig. 2, are shown. Sites indicated with arrows are tissue specific. Those without arrows are not tissue specific. The (A ϩ T) content is indicated and the locations of consensus MAR motifs are shown below. Sequences tested for SATB1 binding met the following criteria: a stretch of A/T/C30 or greater, well mixed, with AT content at least 65%, Ͼ and second stretch of A/T/C20 or greater, with the two stretches not 35 bp apart. In addition, several longer single stretches of at least A/T/C35 were also tested since these may contain a weak binding site. Also shown are possible base unpairing regions, with predicted LFE, as determined by Thermodyn

analysis (see Materials and Methods). This is followed by specific repetitive elements. B, The interruption of a line element with a very GC-rich insertion http://www.jimmunol.org/ is shown with a vertical black oval. sequence defined by one strand consists of exclusively As, Ts and gion for putative GATA-3 sites using either the motif GATA or Cs, excluding Gs, and at least 65% AT content. When the ATC GATC with appropriate 5Ј and 3Ј bases according to Ko and Engel stretches are clustered, it potentially confers high base unpairing (53). We tested eight oligonucleotides for GATA-3 binding by propensity (44). With only one exception, the long ATC sequence EMSA analysis. We focused on oligonucleotide 5 with two tandem stretches within our 19-kb sequence were confined to regions con- GATA-3 binding sites separated by 3 bases, that was located at a taining MARs (Fig. 4). tissue-specific HS site. This oligonucleotide gave a band that was by guest on September 28, 2021

SATB1 binding To test for SATB1 binding to the potential ATC sequence stretches, we performed EMSAs using recombinant GST-SATB1. Ten fragments, ranging in size from 190 to 580 bp, and a control fragment were tested. Two fragments from the regions with strong matrix binding activity were found to bind purified SATB1. One fragment, from plasmid 1231, showed very strong binding (Fig.

5B) with a Kd of 0.04Ð0.15 nM. A fragment from plasmid 1230, bound SATB1 more weakly with a Kd of 1.6Ð2 nM (data not shown). Locations of these SATB1 binding sites are mapped in

Fig. 4. For comparison, the Kd values were 0.3Ð1 nM for in vitro SATB1 binding to six other MAR probes, including fragments from the IgH and ␤ globin MARs, (51) and ranged from 1 to 29 nM for 16 SATB1-binding sequences identified using chromatin immunoprecipitation studies with anti-SATB1 Ab and T cell nu- clear extracts (39). Both SATB1 binding sites are in Thermodyn predicted LFE re- gions, which is consistent with previous findings that SATB1 binds to DNA regions with a high propensity to base unpair (52). In the very high-affinity binding fragment from 1231, there are 3 stretches of FIGURE 5. SATB1 selectively binds with high affinity to a fragment of ATCs of 28, 31, and 27 bases, separated by 8 and 18 bases, respec- the strong MAR of clone 1231. Gel mobility shift assays were performed tively (Fig. 5C). Our other SATB1-binding site had two stretches of with GST-SATB1 protein and, as a negative control, a 190-bp BsaI-BsrGI ATCs of 37 and 33 bases, separated by 33 bp (Fig. 5D). The presence fragment (A) or a contiguous 300-bp BsrGI-EcoRV fragment (B), both of multiple potential SATB1 sites separated by 25 bp or less has been from clone 1231. DNA-binding activity was visualized after electrophore- sis through a 6% acrylamide gel followed by autoradiography. Lanes 1–5 noted in other fragments that bound SATB1 (39). contain 0, 1.2, 0.3, 0.15, and 0.04 nM GST-SATB1 protein. The ATC se- GATA-3 binding quence context from the 1231 MAR fragment which bound SATB1 is shown in C, with the potential SATB1 binding sites, stretches of As, Ts, and Cs, in Another transcription factor that is potentially important for CD8A bold and underlined. The ATC sequence context of the fragment from the is GATA-3. We therefore analyzed the 19-kb re- strong MAR of 1230 which bound SATB1 is shown in D (see text). 3920 HUMAN CD8 CANDIDATE REGULATORY REGION BINDS SATB1 and GATA-3

FIGURE 6. GATA-3 binding to fragments from clones 1231 and 1230. Gel mobility shift assays were performed as described in Materials and Methods with nuclear extracts from stimulated Jurkat cells (a CD4ϩ human T cell line). A, Lane 1, Binding of GATA-3 to oligonucleotide 5 from plasmid 1230 Downloaded from containing two GATA-3 binding sites. The band representing GATA-3 binding is identified with an arrow. Lanes 2 and 3, A supershift with the anti-GATA-3 Ab but not with an isotype control Ab. Competition was performed with self-oligonucleotide, M1 (mutated first site), M2 (mutated second site), M3 (mutated both sites), or an oligonucleotide from the IL-5 promoter containing a GATA-3 binding site (27). The oligonucleotide 5 sequence, with specific mutations indicated in lower case lettering, is CAAAAATTActTCAAGTActTAAAATTTA (GATA-3 motifs bolded (B) Competition of labeled oligonucleotide 5 with other oligonucleotides (see Materials and Methods for sequences) containing putative GATA-3 motifs from the candidate regulatory region. http://www.jimmunol.org/ shifted with an anti-GATA-3 Ab. A mutated oligonucleotide with specific HS cluster, IV, located 3Ј of the last exon, was flanked by the AGATCA site mutation, M1, was able to compete fairly well strong MARs. Further support for the potential importance of these as compared with the wild-type oligonucleotide (Fig. 6A), indicat- regions in CD8 gene expression is the presence of a very strong ing that the M1 site was not as critical for GATA-3 binding as the SATB1 binding site in the MAR linked to HS cluster III and an- AGATAA site which when mutated (M2 mutant) could not com- other site in the 5Ј MAR linked to HS cluster IV. Although we pete. To test the other oligonucleotides, we performed cold com- have discussed these regions separately, they encompass an 11-kb petition studies (Fig. 6B). Oligonucleotide 7, with three potential region that may actually function as a control region-like GATA-3 sites, competed strongly and the others to a lesser extent. regulatory unit. The IL-5 promoter oligonucleotide, which contains a known dou- Positive functional effects by MARs on enhancer activity have by guest on September 28, 2021 ble binding site for GATA-3, competed for binding less well than been observed (56Ð60). Studies have shown that MARs are re- did oligonucleotide 5. This may have to do with the arrangement quired for demethylation of the Ig␬ locus (61, 62) and generation of the two GATA-3 sites in both oligonucleotides. In contrast to of long-range accessibility of chromatin in the Ig␮ locus (15, 63). the tandem GATA-3 sites in oligonucleotide 5, the two GATA-3 An elegant study by Forrester et al. (16) demonstrated that MARs sites in the IL-5 oligonucleotide overlap and are on opposite could facilitate long-range chromatin remodeling. They studied the strands. Interestingly, oligonucleotide 7 is located 400 kb 3Ј of Ig␮ enhancer, which is flanked by MARs, for its ability to activate oligonucleotide 5 within the same MAR. This oligonucleotide, as the VH promoter over a distance of 150 bp or 1.2 kb upstream of well as oligonucleotides 2a, 2b, and 3 showed supershifts with the a methylated or demethylated gene in stable transfectants of B cell anti-GATA-3 Ab (data not shown). lines. They found that the enhancer alone induced local chromatin remodeling, giving rise to a DNase I HS site and local demethyl- Discussion ation, which was sufficient to activate transcription when the Gene transcription is dictated by regulatory elements to which enhancer was 150 bp from the promoter. However, for enhancer- transcription factors bind and by appropriate chromatin modifica- mediated promoter activation over a distance, both MARs were tion (epigenetic regulation). Because chromatin remodeling ap- required for methylated ␮ gene expression. The MARs in combi- pears to be a critical component of gene transcription, DNase I HS nation with the ␮ enhancer could induce acetylation of histones at sites, indicators of relatively open chromatin, are often used as a distal position. This may explain why the ␮ MARs were found signposts for regulatory elements. However, not all HS sites con- to predominantly function in germline transmission but not in tran- tain regulatory elements. Another indicator is colocalization of ma- sient transfection assays where chromatin remodeling does not trix attachment regions with HS sites. For example, there is a sin- need to occur (60). Because the HS sites linked to the strong gle MAR adjacent to both the Ig L chain enhancer, (33) and the MARs that we have identified are located at least 20 kb from either TCR␤ enhancer (54). Also, MARs flank both the IgH (55) and the CD8 promoter, it is possible that the MARs associated with these TCR␦ (56) enhancers. Therefore, identifying regions of the human putative enhancers could promote a similar long-range interaction. CD8 loci that contain HS sites near MARs seemed a logical ap- The higher order chromatin structure that may be required for proach to more rapidly identify strong candidates for regulatory tissue-specific expression of the CD8 genes may in part be regu- elements. lated by the presence of SATB1. SATB1-binding sequences iso- By mapping DNase I HS sites and MARs in the human CD8 lated from a T cell line were localized to the nuclear matrix at the gene complex, we found two regions at the 3Ј end of the CD8B base of chromatin loops in vivo (39). However, in a breast cancer gene in which both HS sites and strong MARs were colocalized. cell line, in which SATB1 is absent, this was not the case for at The tissue-specific DNase I HS cluster III, located between the last least one of these SATB1-binding sequences, indicating that in two CD8␤ exons, was adjacent to a strong MAR. Another tissue- vivo, anchoring of certain MARs onto the nuclear matrix is cell The Journal of Immunology 3921

FIGURE 7. Comparison of DNase I HS sites in the human and mouse CD8 gene complexes. They were aligned using the end of the murine CD8B gene with the homologous human CD8B exon VII.

type specific. The hypothesis was put forth that SATB1 binding to unit composed of multiple HS clusters is likely to be regulating the base of chromatin loops in vivo would create a specific chro- murine CD8A gene expression. matin loop domain structure that was involved in T cell-specific To address the functional significance of potential regulatory gene regulation. The SATB1-binding MARs in the CD8 region elements in the human CD8 gene complex, we have continued to may likewise form a chromatin loop structure that is tissue spe- test in transgenic animals portions from the 95-kb genomic CD8 cific. The very high-affinity SATB1 binding site in the strong fragment that gave tissue-specific expression in transgenic ani- Downloaded from MAR located in the last intron of the CD8B gene and the other site mals. Focusing on the region described in this article, we have in the strong MAR located 4.5 kb downstream could lead to the linked cluster III plus MAR or clusters IV and V plus MARs to a formation of a loop domain that might facilitate CD8 gene expres- genomic human CD8A gene marker gene and analyzed for expres- sion, possibly through long-range histone acetylation of the CD8 sion in transgenic animals. High level expression in a small per- gene. Because of the differences in affinity for SATB1 between the centage of the murine CD8 T cells was noted with both constructs two sites, the formation of specific loop structures may vary de- in all transgenic lines (two to five lines per transgene, our unpub- http://www.jimmunol.org/ pending on the concentration of SATB1. lished data). However, our failure to obtain expression in most of The other protein that we found to bind to the candidate regu- the murine CD8 T cells may be related to the fact that both regions latory region, GATA-3, is also likely to affect CD8A gene expres- together may be required for large numbers of cells expressing the sion. In the mouse a region in the murine HS cluster II located 4Ð5 transgene. Such studies are currently in progress. kb upstream of the CD8A gene (32) contains two GATA-3 binding sites which function in in vitro assays. These GATA-3 binding Acknowledgments sites are within the murine CD8 gene thymocyte-specific enhancer We thank Dr. Carol Webb for the MAR assay protocol. We are very grate- which also contains a SATB1-binding MAR (64). Interestingly,

ful to Dr. Anuradha Ray for the GATA-3 EMSA protocol, nuclear extracts, by guest on September 28, 2021 GATA-3 levels are high in CD4/CD8 thymocytes and then decline and helpful discussions. We also thank Lei Yan and Sarah Cho for tech- as they mature (65). Therefore, GATA-3 may be most important nical assistance and Roberta O’Brien for secretarial assistance. for CD8 expression in the double-positive T cell stage and would bind to the thymocyte-specific enhancer. The strong GATA-3 binding site that we found in the human CD8 tissue-specificHS References 1. Fung-Leung, W., M. W. Schilham, A. Rahemtulla, T. M. Kundig, cluster IV is potentially a functional site in that it has two GATA M. Vollenweider, J. Potter, W. van Ewijk, and T. W. Mak. 1991. CD8 is needed motifs 3 bp apart; double GATA motifs are often found in func- for development of cytotoxic T cells but not helper T cells. Cell 65:443. tional GATA sites. 2. Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, Although it would be very informative to be able to compare the R. M. Zinkernagel, et al. 1991. Normal development and function of CD8ϩ cells location of HS sites between the two species, exact comparisons but markedly decreased helper cell activity in mice lacking CD4. Nature 353:180. are not possible because the human gene complex has ϳ20 kb 3. Devine, L., L. J. Kieffer, V. Aitken, and P. B. Kavathas. 2000. Human CD8␤, but not mouse CD8␤, can be expressed in the absence of CD8␣ as a ␤␤ homodimer. more DNA in the intergenic region (56 kb) compared with the J. Immunol. 164:833. murine region (36 kb), and the murine CD8B gene lacks exons 4. Kober, G., A. L. Griscelli, T. Hercend, and S. C. Meuer. 1991. Expression of VIII and IX. Despite this caveat, some sites may be comparable different CD8 isoforms on distinct human lymphocyte subpopulations. Eur. J. Im- munol. 21:1793. (Fig. 7). For instance, the murine HS cluster IV at the end of the 5. Jarry, A., N. Cerf-Bensussan, N. Brouse, F. Selz, and D. Guy-Grand. 1990. Sub- murine CD8B gene corresponds to our cluster II. In transgenic sets of CD3ϩ (T cell receptor ␣␤ or ␥␦) and CD3Ϫ lymphocytes isolated from animal studies, this region contained regulatory element(s) which normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood. Eur. J. Immunol. 20:1097. directed expression to double-positive thymocytes and mature ϩ 6. Lefrancois, L. 1991. Phenotypic complexity of intraepithelial lymphocytes of the CD8 T cells, but not to CD8␣␣ IEL (9, 12, 13). small intestine. J. Immunol. 146:1746. Another murine regulatory region, associated with DNase HS 7. Kieffer, L. J., L. Yan, J. H. Hanke, and P. B. Kavathas. 1997. Appropriate de- ϳ velopmental expression of human CD8␤ in transgenic mice. J. Immunol. 159: cluster III, located in the intergenic region 16 kb upstream of the 4907. murine CD8A gene, contained an enhancer that was specific for 8. Hostert, A., M. Tolaini, R. Festenstein, L. McNeill, B. Malissen, O. Williams, mature CD8ϩ T cells and CD8␣␣ IEL (10, 11). A fragment R. Zamoyska, and D. Kioussis. 1997. A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice. J. Immunol. 158:4270. from murine DNase HS cluster II, just upstream of the murine 9. Hostert, A., A. Garefalaki, G. Mavria, M. Tolaini, K. Roderick, T. Norton, CD8A gene, when analyzed alone did not display enhancer ac- P. J. Mee, V. L. Tybulewicz, M. Coles, and D. Kioussis. 1998. Hierarchical tivity, but did direct expression to double-positive thymocytes interactions of control elements determine CD8␣ gene expression in subsets of thymocytes and peripheral T cells. Immunity 9:497. when combined with cluster III (9). The location of human 10. Ellmeier, W., M. J. Sunshine, K. Losos, F. Hatam, and D. R. Littman. 1997. An DNase HS clusters III, IV, and V in the intergenic region may enhancer that directs lineage-specific expression of CD8 in positively selected be similar to these murine HS clusters in the intergenic region thymocytes and mature T cells. Immunity 7:537. 11. Hostert, A., M. Tolaini, K. Roderick, N. Harker, T. Norton, and D. Kioussis. (8). The finding that one of the murine clusters did not function 1997. A region in the CD8 gene locus that directs expression to the mature CD8 unless linked to another cluster indicates that a large regulatory T cell subset in transgenic mice. Immunity 7:525. 3922 HUMAN CD8 CANDIDATE REGULATORY REGION BINDS SATB1 and GATA-3

12. Ellmeier, W., M. J. Sunshine, K. Losos, and D. R. Littman. 1998. Multiple de- 39. de Belle, I., S. Cai, and T. Kohwi-Shigematsu. 1998. The genomic sequences velopmental stage-specific enhancers regulate CD8 expression in developing thy- bound to special AT-rich sequence-binding protein 1 (SATB1) in vivo in Jurkat mocytes and in thymus-independent T cells. Immunity 9:485. T cells are tightly associated with the nuclear matrix at the bases of the chromatin 13. Zhang, X., R. Seong, R. Piracha, M. Larijani, M. Heeney, J. R. Parnes, and loops. J. Cell Biol. 141:335. J. W. Chamberlain. 1998. Distinct stage-specific cis-active transcriptional mech- 40. Hambor, J. E., J. Mennone, M. E. Coon, J. H. Hanke, and P. Kavathas. 1993. anisms control expression of T cell coreceptor CD8␣ at double- and single- Identification and characterization of an Alu-containing, T cell-specific enhancer positive stages of thymic development. J. Immunol. 161:2254. located in the last intron of the human CD8␣ gene. Mol. Cell. Biol. 13:7056. 14. Scheuermann, R. H., and W. T. Garrard. 1999. MARs of the antigen receptor and 41. Duret, L., D. Mouchiroud, and C. Gautier. 1995. Statistical analysis of vertebrate C0-receptor genes. Crit. Rev. Eukaryotic Gene Expression 9:295. sequences reveals that long genes are scarce in GC-rich isochores. J. Mol. Evol. 15. Fernandez, L. A., M. Winkler, and R. Grosschedl. 2001. Matrix attachment re- 40:308. gion-dependent function of the immunoglobulin ␮ enhancer involves histone 42. McVean, G. T., L. D. Hurst, and T. Moore. 1996. Genomic evolution in mice and acetylation at a distance without changes in enhancer occupancy. Mol. Cell. Biol. men: imprinted genes have little intronic content. BioEssays 18:773. 21:196. 43. Bode, J., M. Stengert-Iber, V. Kay, T. Schlake, and A. Dietz-Pfeilstetter. 1996. 16. Forrester, W. C., L. A. Fernandez, and R. Grosschedl. 1999. Nuclear matrix Scaffold/matrix-attached regions: topological switches with multiple regulatory attachment regions antagonize methylation-dependent repression of long-range functions. Crit. Rev. Eukaryotic Gene Expression 6:115. enhancer-promoter interactions. Genes Dev. 13:3003. 44. Bode, J., Y. Kohwi, L. Dickinson, T. Joh, D. Klehr, C. Mielke, and 17. Dickinson, L. A., T. Joh, Y. Kohwi, and T. Kohwi-Shigematsu. 1992. A tissue- T. Kohwi-Shigematsu. 1992. Biological significance of unwinding capability of specific MAR/SAR DNA-binding protein with unusual binding site recognition. nuclear matrix-associating DNAs. Science 255:195. Cell 70:631. 45. Kohwi-Shigematsu, T., and Y. Kohwi. 1990. Torsional stress stabilizes extended 18. Alvarez, J. D., D. H. Yasui, H. Niida, T. Joh, D. Y. Loh, and base unpairing in suppressor sites flanking immunoglobulin heavy chain en- T. Kohwi-Shigematsu. 2000. The MAR-binding protein SATB1 orchestrates hancer. Biochemistry 29:9551. temporal and spatial expression of multiple genes during T-cell development. 46. Benham, C., T. Kohwi-Shigematsu, and J. Bode. 1997. Stress-induced duplex Genes Dev. 14:521. DNA destabilization in scaffold/matrix attachment regions. J. Mol. Biol. 274:181. 19. George, K. M., M. W. Leonard, M. E. Roth, K. H. Lieuw, D. Kioussis, F. Grosveld, and J. D. Engel. 1994. Embryonic expression and cloning of the 47. Boulikas, T. 1993. Nature of DNA sequences at the attachment regions of genes murine GATA-3 gene. Development 120:2673. to the nuclear matrix. J. Cell. Biochem. 52:14. Downloaded from 20. Hattori, N., H. Kawamoto, S. Fujimoto, K. Kuno, and Y. Katsura. 1996. Involve- 48. Boulikas, T. 1995. Chromatin domains and prediction of MAR sequences. Int. ment of transcription factors TCF-1 and GATA-3 in the initiation of the earliest Rev. Cytol. 162A:279. step of T cell development in the thymus. J. Exp. Med. 184:1137. 49. Gasser, S. M., B. B. Amati, M. E. Cardenas, and J. F. Hofmann. 1989. Studies on 21. Oosterwegel, M., J. Timmerman, J. Leiden, and H. Clevers. 1992. Expression of scaffold attachment sites and their relation to genome function. Int. Rev. Cytol. GATA-3 during lymphocyte differentiation and mouse embryogenesis. Dev. Im- 119:57. munol. 3:1. 50. Nakagomi, K., Y. Kohwi, L. A. Dickinson, and T. Kohwi-Shigematsu. 1994. A 22. Pandolfi, P. P., M. E. Roth, A. Karis, M. W. Leonard, E. Dzierzak, novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein

F. G. Grosveld, J. D. Engel, and M. H. Lindenbaum. 1995. Targeted disruption SATB1. Mol. Cell. Biol. 14:1852. http://www.jimmunol.org/ of the GATA3 gene causes severe abnormalities in the nervous system and in fetal 51. Dickinson, L. A., and T. Kohwi-Shigematsu. 1995. Nucleolin is a matrix attach- liver haematopoiesis. Nat. Genet. 11:40. ment region DNA-binding protein that specifically recognizes a region with high 23. Ting, C. N., M. C. Olson, K. P. Barton, and J. M. Leiden. 1996. Transcription base-unpairing potential. Mol. Cell. Biol. 15:456. factor GATA-3 is required for development of the T-cell lineage. Nature 384: 52. Dickinson, L. A., C. D. Dickinson, and T. Kohwi-Shigematsu. 1997. An atypical 474. homeodomain in SATB1 promotes specific recognition of the key structural el- 24. Lee, H. J., A. O’Garra, K. Arai, and N. Arai. 1998. Characterization of cis- ement in a matrix attachment region. J. Biol. Chem. 272:11463. regulatory elements and nuclear factors conferring Th2-specific expression of the 53. Ko, L. J., and J. D. Engel. 1993. DNA-binding specificities of the GATA tran- IL-5 gene: a role for a GATA-binding protein. J. Immunol. 160:2343. scription factor family. Mol. Cell. Biol. 13:4011. 25. Leiden, J. M. 1993. Transcriptional regulation of T cell receptor genes. Annu. 54. Chattopadhyay, S., C. E. Whitehurst, and J. Chen. 1998. A nuclear matrix at- Rev. Immunol. 11:539. tachment region upstream of the T cell receptor ␤ gene enhancer binds Cux/CDP 26. Penix, L., W. M. Weaver, Y. Pang, H. A. Young, and C. B. Wilson. 1993. Two and SATB1 and modulates enhancer-dependent reporter gene expression but not essential regulatory elements in the human interferon ␥ promoter confer activa- endogenous gene expression. J. Biol. Chem. 273:29838. by guest on September 28, 2021 tion specific expression in T cells. J. Exp. Med. 178:1483. 55. Cockerill, P. N., M. H. Yuen, and W. T. Garrard. 1987. The enhancer of the 27. Siegel, M. D., D.-H. Zhang, P. Ray, and A. Ray. 1995. Activation of the inter- immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop leukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and anchorage elements. J. Biol. Chem. 262:5394. CLEO elements. J. Biol. Chem. 270:24548. 56. Zhong, X. P., J. Carabana, and M. S. Krangel. 1999. Flanking nuclear matrix 28. Wurster, A. L., G. Siu, J. M. Leiden, and S. M. Hedrick. 1994. Elf-1 binds to a attachment regions synergize with the T cell receptor ␦ enhancer to promote critical element in a second CD4 enhancer. Mol. Cell. Biol. 14:6452. V(D)J recombination. Proc. Natl. Acad. Sci. USA 96:11970. 29. Yamagata, T., K. Mitani, H. Ueno, Y. Kanda, Y. Yazaki, and H. Hirai. 1997. 57. Blasquez, V. C., M. Xu, S. C. Moses, and W. T. Garrard. 1989. Immunoglobulin Triple synergism of human T-lymphotropic virus type 1-encoded tax, GATA- ␬ gene expression after stable integration. I. Role of the intronic MAR and en- binding protein, and AP-1 is required for constitutive expression of the interleu- hancer in plasmacytoma cells. J. Biol. Chem. 264:21183. kin-5 gene in adult T-cell leukemia cells. Mol. Cell. Biol. 17:4272. 58. Goyenechea, B., N. Klix, J. Yelamos, G. T. Williams, A. Riddell, 30. Yamagata, T., J. Nishida, R. Sakai, T. Tanaka, H. Honda, N. Hirano, H. Mano, M. S. Neuberger, and C. Milstein. 1997. Cells strongly expressing Ig␬ transgenes Y. Yazaki, and H. Hirai. 1995. Of the GATA-binding proteins, only GATA-4 show clonal recruitment of hypermutation: a role for both MAR and the enhanc- selectively regulates the human interleukin-5 gene promoter in interleukin-5- ers. EMBO J. 16:3987. producing cells which express multiple GATA-binding proteins. Mol. Cell. Biol. 59. Xu, M., R. E. Hammer, V. C. Blasquez, S. L. Jones, and W. T. Garrard. 1989. 15:3830. Immunoglobulin ␬ gene expression after stable integration. II. Role of the in- 31. Zhang, D. H., L. Yang, and A. Ray. 1998. Differential responsiveness of the IL-5 tronic MAR and enhancer in transgenic mice. J. Biol. Chem. 264:21190. and IL-4 genes to transcription factor GATA-3. J. Immunol. 161:3817. 60. Forrester, W. C., C. van Genderen, T. Jenuwein, and R. Grosschedl. 1994. De- 32. Landry, D. B., J. D. Engel, and R. Sen. 1993. Functional GATA-3 binding sites ␮ within murine CD8 ␣ upstream regulatory sequences. J. Exp. Med. 178:941. pendence of enhancer-mediated transcription of the immunoglobulin gene on 33. Cockerill, P. N., and W. T. Garrard. 1986. Chromosomal loop anchorage of the nuclear matrix attachment regions. Science 265:1221. ␬ immunoglobulin gene occurs next to the enhancer in a region containing to- 61. Kirillov, A., B. Kistler, R. Mostoslavsky, H. Cedar, T. Wirth, and Y. Bergman. ␬ ␬ poisomerase II sites. Cell 44:273. 1996. A role for nuclear NF- B in B-cell-specific demethylation of the Ig locus. 34. Webb, C. F., C. Das, K. L. Eneff, and P. W. Tucker. 1991. Identification of a Nat. Genet. 13:435. matrix-associated region 5Ј of an immunoglobulin heavy chain variable region 62. Lichtenstein, M., G. Keini, H. Cedar, and Y. Bergman. 1994. B cell-specific gene. Mol. Cell. Biol. 11:5206. demethylation: a novel role for the intronic ␬ chain enhancer sequence. Cell 35. Greally, J. M., T. A. Gray, J. M. Gabriel, L. Song, S. Zemel, and R. D. Nicholls. 76:913. 1999. Conserved characteristics of heterochromatin-forming DNA at the 15q11Ð 63. Jenuwein, T., W. C. Forrester, L. A. Fernandez-Herrero, G. Laible, M. Dull, and q13 imprinting center. Proc. Natl. Acad. Sci. USA 96:14430. R. Grosschedl. 1997. Extension of chromatin accessibility by nuclear matrix at- 36. Jong, M. T., T. A. Gray, Y. Ji, C. C. Glenn, S. Saitoh, D. J. Driscoll, and tachment regions. Nature 385:269. R. D. Nicholls. 1999. A novel imprinted gene, encoding a RING zinc-finger 64. Banan, M., I. C. Rojas, W.-H. Lee, H. L. King, J. V. Harriss, R. Kobayashi, protein, and overlapping antisense transcript in the Prader-Willi syndrome critical C. F. Webb, and P. D. Gottlieb. 1997. Interaction of the nuclear matrix-associated region. Hum. Mol. Genet. 8:783. region (MAR)-binding proteins, SATB1 and CDP/Cux, with a MAR element 37. Greally, J. M., M. E. Guinness, J. McGrath, and S. Zemel. 1997. Matrix-attach- (L2a) in an upstream regulatory region of the mouse CD8␣ gene. J. Biol. Chem. ment regions in the mouse 7F imprinted domain. Mamm. Genome 272:18440. 8:805. 65. Nawijn, M. C., R. Ferreira, G. M. Dingjan, O. Kahre, D. Drabek, A. Karis, 38. Kramer, J. A., G. B. Singh, and S. A. Krawetz. 1996. Computer-assisted search F. Grosveld, and R. W. Hendriks. 2001. Enforced expression of GATA-3 during for sites of nuclear matrix attachment. [Published erratum appears in 1996 T cell development inhibits maturation of CD8 single-positive cells and induces Genomics 35:273.] Genomics 32:305. thymic lymphoma in transgenic mice. J. Immunol. 167:715.