The genomic sequences bound to special AT-rich sequence-binding protein 1 (SATB1) in vivo in Jurkat T cells are tightly associated with the nuclear matrix at the bases of the chromatin loops

Abstract

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1-1. 1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island-containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear "halos" with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type-specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell-specific gene regulation.

Figure 1

SATB1 is expressed in Jurkat cells and is a component of the nuclear matrix. (A) Western blot with anti-SATB1 antibody from Jurkat cell extracts. Cell extracts from Jurkat cells were either untreated (lane 1) or immunoprecipitated with preimmune (lane 2) or anti-SATB1 antibody (lane 3). The protein extracts and immunoprecipitates were then resolved by 10% SDS-PAGE and electrotransferred to an immobilon-P membrane. The membrane was then probed using anti-SATB1 antibody and was visualized using an HRP-conjugated secondary antibody followed by ECL detection. Bio-Rad biotinylated molecular mass markers are indicated on the left, and the position of SATB1 migration is shown by an arrow on the right. (B) Subcellular localization of SATB1. Mouse thymocytes (5 × 10⁶) were fractionated as described in Materials and Methods: Lane 1, supernatant of 0.5% Triton X-100–CSK extraction (soluble fraction); lane 2, the “cytoskeleton” fraction; lane 3, the “chromatin” fraction; lane 4, the nuclear matrix fraction as described in Materials and Methods.

Figure 2

Outline of in vivo cross-linking and binding site cloning protocol. The general scheme for the cross-linking of DNA and proteins and subsequent isolation and cloning of SATB1-binding sites is shown. For a detailed description of the protocol see the Materials and Methods section.

Figure 3

Immunoprecipitation of SATB1 and urea gradient purification of genomic DNA. (A) In vitro cross-linking of GST– SATB1 to a radiolabeled synthetic MAR. The GST–SATB1 protein was mixed with a radiolabeled MAR probe representing a concatemerized IgH enhancer MAR. After exposure to 1% formaldehyde for the indicated period of time, samples were immunoprecipitated with either preimmune (solid histograms) or anti-SATB1 antibody (hatched histograms). The cpm associated with the immunoprecipitates was determined by liquid scintillation counting. (B) Urea gradient purification of genomic DNA. Jurkat cells were grown in the presence of [³H]thymidine and [¹⁴C]leucine for a period of 24 h. Cells were then either untreated (top) or subjected to in vivo formaldehyde cross-linking for a period of 2 h (bottom). Cells were lysed and loaded onto 5–8 M urea gradients and centrifuged as described in the Methods section. After centrifugation, the gradients were fractionated and ³H and ¹⁴C counts were determined by liquid scintillation counting. As a separate experiment, similar urea gradient centrifugation was performed with Jurkat cells grown in the presence of [³H]uridine to monitor RNA distribution in the gradient (top).

Figure 3

Immunoprecipitation of SATB1 and urea gradient purification of genomic DNA. (A) In vitro cross-linking of GST– SATB1 to a radiolabeled synthetic MAR. The GST–SATB1 protein was mixed with a radiolabeled MAR probe representing a concatemerized IgH enhancer MAR. After exposure to 1% formaldehyde for the indicated period of time, samples were immunoprecipitated with either preimmune (solid histograms) or anti-SATB1 antibody (hatched histograms). The cpm associated with the immunoprecipitates was determined by liquid scintillation counting. (B) Urea gradient purification of genomic DNA. Jurkat cells were grown in the presence of [³H]thymidine and [¹⁴C]leucine for a period of 24 h. Cells were then either untreated (top) or subjected to in vivo formaldehyde cross-linking for a period of 2 h (bottom). Cells were lysed and loaded onto 5–8 M urea gradients and centrifuged as described in the Methods section. After centrifugation, the gradients were fractionated and ³H and ¹⁴C counts were determined by liquid scintillation counting. As a separate experiment, similar urea gradient centrifugation was performed with Jurkat cells grown in the presence of [³H]uridine to monitor RNA distribution in the gradient (top).

Figure 4

SATB1 binds to genomic DNA in vivo. (A) PCR amplification of immunoprecipitated DNA. After purification of immunoprecipitated DNA and linker ligation, PCR amplification was performed as described in the Materials and Methods section using 17 cycles. PCR products from preimmune (lane 1) and anti-SATB1 immunoprecipitated DNA (lane 2) were examined by 1% agarose gel electrophoresis followed by staining with ethidium bromide. Lane M, 1-kb molecular size markers. (B) Gel mobility shift with anti-SATB1 immunoprecipitated DNA. PCR products shown by a bracket in A were isolated from the gel and cloned into pBluescript. Insert DNA in different size ranges was tested for SATB1-binding activity. DNA in the range of 0.6 kb (lanes 1–3) and 1-kb ranges (lanes 4–6) are shown. Lanes 1 and 4 contain 0 nM; lanes 2 and 5, 8 nM; lanes 3 and 6, 16 nM GST– SATB1 protein. (C) PCR amplification of preimmune immunoprecipitated DNA. PCR products were generated using 35 cycles of amplification, and the resulting DNA was visualized in 1% agarose gel stained with ethidium bromide (lane 1). The region shown by a bracket was cloned into pBluescript. Lane M, 1-kb molecular size markers. (D) Gel mobility shift assay with preimmune immunoprecipitated DNA. Inserts derived from DNA shown by the bracket from C were isolated and used in a gel mobility shift assay with GST–SATB1 protein. Lanes 1–3 contain 0, 8, and 16 nM GST–SATB1 protein, respectively. (E) Gel mobility shift assay with precleared anti-SATB1 antibody. A similar experiment as described for C and D was performed with precleared anti-SATB1 antiserum, which was prepared by adding SATB1 to the serum, followed by immunoprecipitation. After a 35-cycle amplification, the cloned DNA failed to demonstrate a gel shift with GST–SATB1. Lanes 1–3 contain 0, 8, and 16 nM GST–SATB1, respectively.

Figure 5

In vivo SATB1-binding sites contain ATC sequences. The nucleotide sequence of two of the cloned SATB1-binding sites, SBS-3 and SBS-14, is shown. The presence of ATC/ATG sequences are indicated by underlining or a dotted line. These clones are representative, and all sequences contained similar ATC sequences. In SBS-14, the central region with an ATC sequence stretch juxtaposed with surrounding ATC sequence stretches is likely to be a preferential site for SATB1 binding.

Figure 6

SATB1 binds specifically and with high affinity to cloned binding sites. Gel mobility assay was performed with cloned in vivo SATB1-binding sequences SBS-3 (A) and SBS-14 (B) as well as synthetic MAR wild-type (25)₅ (C) and GST– SATB1 protein. DNA binding activity was visualized after electrophoresis through a 6% acrylamide gel followed by autoradiography. In the left panels, lanes 1–6 contain 0, 0.5, 1, 2, 4, and 8 nM GST–SATB1 protein. The right panel shows a gel mobility shift competition using a 50-fold molar excess of unlabeled wild-type (25)₅ (wt) or a nonbinding mutated version mutated (24)₈ (mut). The concentration of SATB1 used was 4 nM.

Figure 7

SATB1-binding sites are localized to the base of chromatin loops. In situ hybridization with Jurkat nuclei (top), nuclear halo (middle), and nuclear matrix (bottom) was performed using in vivo SATB1-binding sequences, SBS-2, SBS-3, and SBS-11, and control DNA, pTP18 (for U2 snRNA) and pH5SB (for the 5S rRNA gene), as DNA probes. These probes were labeled with biotin-11-dATP. Total DNA was stained with propidium iodide (red), and specific sequence hybridization was detected with FITC-conjugated extravidin (yellow-green). Since most of the genomic DNA has been removed from the nuclear matrix preparations, no propidium iodide staining was detectable in the nuclear matrix. In situ hybridization signals to the 5S rRNA within the distended chromatin halo are indicated by a white arrow.

Figure 8

Anchoring of a SATB1-binding genomic sequence to the nuclear matrix is cell type dependent. In situ hybridization with SBS-11 was performed on nuclei, halos, and nuclear matrices prepared from Jurkat cells and breast cancer SK-BR-3 cells. The detection method was identical to that described in the legend to Fig. 7 for nuclei and the nuclear matrix. To enhance visualization of hybridization signals within the nuclear halos, the hybridization signal was detected with rhodamine-conjugated extravidin (red) over total DNA staining with DAPI (blue). The sizes of nuclei, halos, and nuclear matrix from SK-BR-3 are in general larger than those of Jurkat cells as shown. The exposure time was extended for the SK-BR-3 halos to 30 s (15 s for the Jurkat halos) to reproduce all rhodamine signal detected within the halo region in the final photograph. Because of the release of the sequences into the distended loop region, the SBS-11 sequence apparently became more accessible to hybridization with the DNA probe. With a 30-s exposure, signals in the Jurkat halo preparations became more intense but remained condensed in the residual nucleus (data not shown). SK-BR-3 cells are aneuploid and giving more hybridization spots on nuclei.