Promoter-specific targeting of human SWI-SNF complex by Epstein-Barr virus nuclear protein 2 - PubMed (original) (raw)

Promoter-specific targeting of human SWI-SNF complex by Epstein-Barr virus nuclear protein 2

D Y Wu et al. J Virol. 2000 Oct.

Abstract

The multiprotein human SWI-SNF (hSWI-SNF) complex is a chromatin-remodeling machine that facilitates transcription by overcoming chromatin-mediated gene repression. We had previously shown that hSNF5/INI1, an intrinsic, consistent component of the hSWI/SNF complex, is associated with Epstein-Barr nuclear antigen 2 (EBNA2) and have proposed that EBNA2 directs this complex to key EBNA2-responsive viral and cellular genes. Using chromatin immunoprecipitation and quantitative PCR, we show that antibodies directed against components of the hSWI-SNF complex preferentially precipitate chromatin-associated DNA that contains a targeted EBNA2-responsive element in the context of both episomal and cellular chromatin. This enrichment does not occur in EBNA2-negative cells or when the EBNA2-responsive element is mutated. The stable association of the hSWI-SNF complex with the EBNA2-responsive promoter can also be disrupted by deletion of the TATA element, suggesting that EBNA2 in itself is insufficient to mediate stable targeting of the hSWI-SNF complex. These results demonstrate that recruitment of the hSWI-SNF complex to selected promoters can occur in vivo through its interaction with site-specific activator proteins and that stable targeting may require the presence of basal transcription factors.

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Figures

FIG. 1

Schematic representation of pTpTATAMG and micrococcal nuclease digestion of pTpTATAMG and pTpmutTATAMG. (A) Main features of plasmid pTpTATAMG. The sequence of the EBV TP1/LMP2A promoter, TATA box, upstream Myc-globin reporter gene, and heptameric RBP-Jκ/CBF1 binding sites within the TP1/LMP2A promoter are indicated. Substitutions in plasmid pTpmutTATAMG (mut) replace the GGG sequence within the RPB-Jκ site with TTT. (B) Micrococcal nuclease (Mnase) digestion and indirect end labeling of the upstream region of the Myc-globin reporter gene. The probe for hybridization was derived from the 237-bp _Nsi_I/_Bam_HI fragment of pTpTATAMG (position indicated). The eight micrococcal nuclease-hypersensitive sites identified are numbered 1 to 8. Proposed positions of the nucleosomes (circles) are depicted in the lower panel. Marker lanes (1 and 2) contain 10 and 2 pg of pTpTATAMG digested with _Not_I and _Nsi_I.

FIG. 2

Purification of chromatin fragments. (A) Aliquots of DNA extracted from chromatin fractions derived from sucrose gradient purification were separated on a 1.5% agarose gel and stained with ethidium bromide. Migration of mono- and dinucleosomes is indicated at the right. Lane B designates the bottom gradient fraction, which includes chromatin fragment sediments; lanes M contain size markers. (B) Immunoprecipitation of sucrose gradient fractions with anti-hSNF5/INI1 antibodies. 35S-labeled chromatin fragments were prepared and purified on the sucrose gradient as described for panel A. Each sucrose gradient fraction was then immunoprecipitated with Rb2464 (anti-hSNF5 antibody), separated by SDS-PAGE, and visualized by autoradiography.

FIG. 3

Quantitative PCR of DNA from immunoprecipitated chromatin fractions. Sucrose gradient-purified chromatin fragments pooled from fractions 8 to 13 were immunoprecipitated with preimmune serum (Preimmune), anti-hSNF5 antibody (α-hSNF5), or anti-BRG1 antibody (α-BRG1). DNA isolated from immunoprecipitates was subjected to quantitative PCR to determine relative abundance of the TP1/LMP2A promoter and β-lactamase sequences. Control PCR series were performed with plasmid pTpTATAMG (10−14 to 10−11 M) as the template. The results were analyzed on a PhosphorImager and are reported as the relative amount of PCR product compared with preimmune control (indicated below of each lane). (A) Autoradiogram of quantitative PCR of Myc-globin sequences from Raji/pTpTATAMG (Wt [wild type]; lanes 8 to 10) and Raji/pTpmutTATAMG (Mut [mutant]; lanes 5 to 7) chromatin fragments immunopurified with preimmune or hSNF5- or BRG1-specific antibodies. (B) Autoradiogram of PCR of β-lactamase sequence from immunopurified Raji/pTpTATAMG (Wt; lanes 7 and 8) and Raji/ pTpmutTATAMG (Mut; lanes 5 and 6) chromatin fragments using the AmpR primer set. (C) Autoradiogram of PCR of Myc-globin sequence from chromatin immunoprecipitation of BL41/B95 (EBNA2-positive) or BL41/P3HR1 (EBNA2-negative) cells that harbor the episomal pTpTATAMG.

FIG. 4

Analysis of the contribution of TP1/LMP2A promoter and TATA elements to reporter expression and targeting of the hSWI-SNF complex. (A) The steady-state levels of Myc-globin and GAPDH mRNA of Raji/pTpTATAMG, Raji/pTpmutTATAMG, Raji/pTATAMG, and Raji/pTpMG cells were determined by the S1 nuclease assay. The results were normalized for the relative copy number of reporter episomes present in these cells (4.8 for pTATAMG, 4.0 for pTpMG, 0.5 for pTpmutTATAMG, and 1.0 for pTpTATAMG) and reported as relative mRNA levels with respect to the mRNA in the promoterless Raji/pTATAMG cells. (B) Targeting of hSNF5/INI1, TBP, and Pol II was determined by immunoprecipitation and PCR incorporation of [α-32P]dATP. Input DNA prior to immunoprecipitation was quantitated by PCR for both Myc-globin and β-lactamase (AmpR) sequences, showing nearly equal input DNA in cells harboring the four episomal constructs. Quantitative incorporation of [α-32P]dATP is linear over 2 orders of magnitude of template concentrations for both AmpR and MycGb primer sets (lower panel).

FIG. 5

Targeting of the hSWI-SNF complex to the CD23 gene. (A) Schematic of the upstream region of the CD23 gene containing exons I, II, and b. The two RBP-Jκ/CBF1 binding sites present in this region are indicated by solid rectangles. The upstream sequence from exon I is shown with the RPB-Jκ/CBF1 and TATA elements. Positions of the PCR primers (sequences shown in Table 1) and their product lengths are also shown. (B) Targeting of the hSWI-SNF complex to three regions on the CD23 gene in BL41/B95 and BL41/P3HR1 cells were measured by immunoprecipitation and quantitative PCR with specific primer sets. The autoradiogram of quantitative PCR using the CD23B primer set is depicted. Lanes 1 to 4, PCR of template controls; lanes 5 and 8, PCR results from the preimmunoprecipitated input (1/5 of total) chromatin; lanes 6, 7, 9, and 10, PCR results from the immunoprecipitated samples. (C) Results of PhosphorImager analysis of PCR performed with CD23A, CD23B, and CD23C primer sets on chromatin immunoprecipitation samples. Results are reported as the percentage of signal obtained from chromatin prior to immunoprecipitation.

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Promoter-specific targeting of human SWI-SNF complex by Epstein-Barr virus nuclear protein 2 - PubMed (original) (raw)