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. 2002 Apr;22(8):2598-606.
doi: 10.1128/MCB.22.8.2598-2606.2002.

Scaffold/matrix attachment region elements interact with a p300-scaffold attachment factor A complex and are bound by acetylated nucleosomes

Joost H A Martens  1 , Matty Verlaan, Eric Kalkhoven, Josephine C Dorsman, Alt Zantema
Affiliations

Scaffold/matrix attachment region elements interact with a p300-scaffold attachment factor A complex and are bound by acetylated nucleosomes

Joost H A Martens et al. Mol Cell Biol. 2002 Apr.

Abstract

The transcriptional coactivator p300 regulates transcription by binding to proteins involved in transcription and by acetylating histones and other proteins. These transcriptional effects are mainly at promoter and enhancer elements. Regulation of transcription also occurs through scaffold/matrix attachment regions (S/MARs), the chromatin regions that bind the nuclear matrix. Here we show that p300 binds to the S/MAR binding protein scaffold attachment factor A (SAF-A), a major constituent of the nuclear matrix. Using chromatin immunoprecipitations, we established that both p300 and SAF-A bind to S/MAR elements in the transiently silent topoisomerase I gene prior to its activation at G(1) during cell cycle. This binding is accompanied by local acetylation of nucleosomes, suggesting that p300-SAF-A interactions at S/MAR elements of nontranscribed genes might poise these genes for transcription.

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Figures

FIG. 1.
FIG. 1.
p300 interacts with SAF-A in vivo and in vitro. (A) Endogenous p300 coimmunoprecipitates with SAF-A. U-2 OS whole-cell extracts were immunoprecipitated with antibodies against p300 (p300-1, p300-3, and N15) or SAF-A and tested on Western blot for coimmunoprecipitated SAF-A or p300. (B) GST pull-down assays. In vitro-translated [35S]methionine-labeled 12S-E1A or SAF-A (aa 633 to 806) retained on GST, GST-p300ADA2 (aa 1573 to 1731), GST-p300CH3 (aa 1726 to 1848), or GST-p300BAIT (aa 1573 to 1848) was visualized by autoradiography. (C) Mapping of the SAF-A-p300 interaction by GST pull-down. In vitro-translated SAF-A subdomains retained on GST-p300CH3 were visualized by autoradiography.(D) GST-SAF-A (aa 537 to 806) retains endogenous p300 from U-2 OS whole-cell extracts as visualized on a Western blot with an anti-p300 antibody. (E) GST pull-down assay. GST-E1A (aa 1 to 90) and GST-p300CH3 retain endogenous p300 or SAF-A, respectively, as visualized on Western blots. (F) Competition assay. Increasing amounts of E1A or BSA were used for competing away in vitro-translated SAF-A retained on GST-p300CH3.
FIG. 2.
FIG. 2.
SAF-A and p300 cofractionate. (A) U-2 OS cell extract was fractionated on a Superose 6 column, and fractions were collected and tested on Western blot for the presence of SAF-A, p300, and the protein kinase p90rsk. At the top the elution of markers is indicated by their molecular sizes in kilodaltons. (B) RNP particles were isolated from whole-cell extracts in the 45% sucrose fractions (lanes 1 and 2 represent two independent gradients). The presence of SAF-A, hnRNP L, histones, and p300 was tested by Western blot. (C) U-2 OS nuclear skeleton was separated by two procedures into a chromatin and an RNP-associating fraction. In the sonication procedure, the pellet (p) represents the RNP-associated fraction and the supernatant (s) represent the chromatin-associated fraction. In the RNase procedure, the pellet represents the chromatin-associated fraction and the supernatant represents the RNP-associated fraction. The presence of p300, SAF-A (120 kDa), and histones was detected by Western blotting.
FIG. 3.
FIG. 3.
SAF-A/p300 bind S/MAR elements. (A) Schematic representation of the topoisomerase I gene (39, 53) showing the MI and MII S/MAR elements. Vertical bars represent the exons, open boxes represent the S/MAR elements, and triangles represent the positions of the internal controls in panel B. (B and C) ChIP assays with U-2 OS cells with antibodies against SAF-A, p300, and acetylated histone H3. Nonimmune serum (ni) was used as a control. The DNAs of the immunoprecipitated fractions were isolated, and topoisomerase I sequences in the immunoprecipitated DNA were amplified by PCR. Asterisks indicate that five times more input was used in these assays. TopMI and TopMII are fragments within the S/MAR elements of topoisomerase I; pTOP is a fragment within the promoter region, and exon 13 is a fragment within exon 13 of the topoisomerase I gene. In panel C, additional S/MAR sequences were tested: MII of the topoisomerase I gene (TopMII) and the S/MAR sequences of the c-myc, Igκ, and β-globin genes, respectively. As a control for specific amplification, a primer set in the c-jun promoter was used. The chromosomal location of the various genes is shown on the right.
FIG. 4.
FIG. 4.
Absence of binding of SAF-A or p300 to S/MAR elements in virally transformed cells. (A) ChIP assays with adenovirus-transformed 911 cells with a control antibody (ni) and antibodies against SAF-A, p300, acetylated histone H3, and ATF-2. Primer sets in the topoisomerase I promoter (pTOP) and the c-jun promoters were used as a control. Asterisks indicate that five times more input was used. (B) Quantification of ChIP results. The relative difference in the amount of precipitated DNA as determined by real-time PCR is presented for the ChIP results found for the MII S/MAR element of topoisomerase I in Fig. 4A (911 cells) and Fig. 3B (U-2 OS cells) on a log scale. Nonimmune values are set at 1. (C) Absence of binding of SAF-A or p300 to S/MAR elements in an SV40-transformed cell line compared to nontransformed VH10 cells. The relative difference inamount of precipitated DNA as determined by real-time PCR is presented for the ChIP results found for the MII S/MAR element of topoisomerase I (TopMII). (D) RT-PCR for the mRNA expression of the topoisomerase I and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) genes in U-2 OS, 911, VH10, and SV40-transformed VH10 cells.
FIG. 5.
FIG. 5.
Inactive topoisomerase I gene has SAF-A, p300, and acetylated histone H3 bound to its S/MAR element. (A) RT-PCR for the RNA expression of the topoisomerase I, p300, and GAPDH genes of serum-arrested T98G cells and after subsequent stimulation by serum for 6, 12, 18, 36, and 72 h, representing growing and subsequently (72 h) density-arrested cells. (B) ChIP assays with the serum-stimulated cells were analyzed with real-time PCR with primers within the topoisomerase I S/MAR element and in the topoisomerase I promoter. Depicted is the relative level of precipitated DNA with antibodies to SAF-A or p300 in comparison to a nonimmune control antibody. (C) ChIP assays with an antibody against acetylated histone H3 show high levels of histone H3 acetylation at S/MAR elements of topoisomerase I when the gene is inactive, in contrast to promoter and exon sequences that only show hyperacetylated histone H3 when the gene is expressed.
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