A Nuclear Matrix Attachment Region Upstream of the T Cell Receptor β Gene Enhancer Binds Cux/CDP and SATB1 and Modulates Enhancer-dependent Reporter Gene Expression but Not Endogenous Gene Expression

Abstract

We have previously identified a DNase I-hypersensitive site in the T cell receptor ␤ locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer E ␤ and is induced during CD4 ؊ CD8 ؊ to CD4 ؉ CD8؉ thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MAR ␤ . These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MAR expressed by an individual T cell (4, 5). After V ␤ D ␤ J ␤ rearrangement, a mature transcript is initiated from the V ␤ promoter in a T cell-specific manner (6). To achieve the lineage-, stage-, and allele-specific TCR␤ gene rearrangement and transcription, many cis-acting elements and their associated transacting factors are likely to be involved (4, 5).To date, the TCR␤ gene enhancer (E ␤ ) is the only cis-regulatory element demonstrated to be required for both the lineage-and stage-specific transcription and rearrangement of the TCR␤ gene (7-12). Although the V ␤ promoter is required for lineage-specific TCR␤ transcription, its role in regulating V ␤ gene rearrangement remains unclear (13-17). In addition to V ␤ promoters and E ␤ , there are likely other cis-regulatory elements involved in the control of various aspects of TCR␤ gene rearrangement and transcription. In particular, nuclear matrix attachment regions (MAR) are a class of cis-regulatory elements found in many genetic loci that are distinct from transcriptional promoters and enhancers, and yet are often closely associated with these regulatory elements (18 -20). MARs are typically AT-rich DNA sequences that bind to the nuclear matrix, often contain topoisomerase II cleavage sites, and exhibit a propensity for base unpairing when subjected to superhelical strain (21, 22). They have been proposed to be involved in transcription, DNA recombination, replication, and repair (23). In the immunoglobulin heavy chain (IgH) locus, MARs flank the intronic enhancer E and are in close proximity to V H promoters (22)(23)(24)(25)(26). Reporter gene assays in cell lines and transgenic mice have suggested that these MARs exert both positive and negative effects on IgH gene transcription and promote long range chromatin accessibility (27-31). A highly conserved MAR is also found 200 base pairs (bp) upstream of the intronic immunoglobulin (Ig) enhancer in mouse, human, and rabbit (22,32). Together, the Ig MAR and enhancer promote demethylation, transcription, recombination, and somatic hypermutation of the locus although no specific function has been attributed to the MAR alone (33-36). The presence of MARs at other antigen receptor loci has not been reported.To characteriz...

Phosphorylation of the CCAAT Displacement Protein (CDP)/Cux Transcription Factor by Cyclin A-Cdk1 Modulates Its DNA Binding Activity in G2

“…Nuclear extracts were prepared according to the procedure of Lee et al (66), except that nuclei were obtained by submitting cells to 3 freeze/ thaw cycles in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol) (25). Whole cell extracts from Sf9 cells were prepared in Buffer M (50 mM Hepes, pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM MgCl 2 , 1% Triton X-100, and protease inhibitor tablet (Roche Molecular Biochemicals)).…”

CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

“…Because CDP is a matrix-associated region-binding protein (7,15,41,64), complete chromatin structure may be important for maximum effects on repressor function. Therefore, analysis of CDP binding site mutants in stable-transfection assays allowed us to analyze integrated templates and to ensure that the majority of cells contained the reporter construct.…”

“…Together, these observations argue that cooperative interactions between different CDP DNA-binding domains and between different CDP molecules bound to adjacent DNA-binding sites may be important for promoter rec-ognition in vivo. The ability of CDP to bind to the promoters and enhancers of a large number of genes, including those for c-mos, ␥-globin, Ncam, histones, gp91-phox, HPV E1, E6, and E7, c-myc, the immunoglobulin heavy and light chains, TCR␤, and CD8␣, MMTV, and the cystic fibrosis transmembrane conductance regulatory gene (1,7,8,15,16,21,31,38,41,57,61,64), may depend upon the presence of multiple DNAbinding domains with different DNA-binding and protein interaction specificities.…”

The Matrix Attachment Region-binding Protein SATB1 Interacts with Multiple Elements within the gp91 Promoter and Is Down-regulated during Myeloid Differentiation

“…Examples include the T cell receptor ␤ gene enhancer (22), the mouse CD8a gene upstream regulatory region (23), and the mouse mammary tumor virus long terminal repeat (24). Unlike many other MAR-binding proteins, SATB1 is a cell type-specific protein and is predominantly expressed in T cells and thymocytes (24 -27).…”

“…However, although both low-mobility complexes exhibit a similar DNAbinding specificity, only the upper complex is disrupted by antiserum directed against CDP. Several regulatory elements in other promoters exhibit a similar doublet of low mobility EMSA complexes in which the upper complex contains CDP and the lower complex contains the MAR-binding protein, SATB1 (22)(23)(24). In addition, the proximal gp91 phox promoter contains several regions of ATC sequence (5), the preferred binding site for SATB1.…”