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SATB1 and GATA-3 and Matrix Attachment Regions Which Bind

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SATB1 and GATA-3 and Matrix Attachment Regions Which Bind 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 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 chromatin 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 protein, 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: 3915–3922. 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 genes, 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␤ (9–13). ‡ 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 50–100 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 proteins 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.
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