SNF5 reexpression in malignant rhabdoid tumors regulates transcription of target genes by recruitment of SWI/SNF complexes and RNAPII to the transcription start site of their promoters

Abstract

Malignant rhabdoid tumor (MRT), a highly aggressive cancer of young children, displays inactivation or loss of the hSNF5/INI1/SMARCB1 gene, a core subunit of the SWI/SNF chromatin-remodeling complex, in primary tumors and cell lines. We have previously reported that reexpression of hSNF5 in some MRT cell lines causes a G1 arrest via p21(CIP1/WAF1) (p21) mRNA induction in a p53-independent manner. However, the mechanism(s) by which hSNF5 reexpression activates gene transcription remains unclear. We initially searched for other hSNF5 target genes by asking whether hSNF5 loss altered regulation of other consensus p53 target genes. Our studies show that hSNF5 regulates only a subset of p53 target genes, including p21 and NOXA, in MRT cell lines. We also show that hSNF5 reexpression modulates SWI/SNF complex levels at the transcription start site (TSS) at both loci and leads to activation of transcription initiation through recruitment of RNA polymerase II (RNAPII) accompanied by H3K4 and H3K36 modifications. Furthermore, our results show lower NOXA expression in MRT cell lines compared with other human tumor cell lines, suggesting that hSNF5 loss may alter the expression of this important apoptotic gene. Thus, one mechanism for MRT development after hSNF5 loss may rely on reduced chromatin-remodeling activity of the SWI/SNF complex at the TSS of critical gene promoters. Furthermore, because we observe growth inhibition after NOXA expression in MRT cells, the NOXA pathway may provide a novel target with clinical relevancy for treatment of this aggressive disease.

Figure 1

hSNF5-induced p53 target genes’ expression. RNA was extracted at 24 hours after infection with Ad-hSNF5 and Ad-GFP. The mRNA levels were measured for each gene by real-time qRT-PCR and normalized for β-actin expression and relative to the Ad-GFP for each genes. Values are the mean of 3 independent experiments; bars, ± SD.

Figure 2

hSNF5-induced NOXA mRNA expression. A, RNA was extracted at the indicated times after infection with Ad-hSNF5 and Ad-GFP. The mRNA levels were measured for each gene by real-time qRT-PCR and normalized for β-actin expression. Values are the mean of 3 independent experiments; bars, ±SD; un, uninfected control. B, NOXA mRNA expression in 6 MRT cell lines (A204.1, TTC642, G401.6TG, TTC549, TM87-16, and TTC549) by real-time qRT-PCR. The MCF7 cell line was used as a control. The NOXA mRNA levels were measured for each gene by real-time qRT-PCR and normalized for β-actin expression. Values are the mean of 3 independent experiments; bars, ±SD.

Figure 3

hSNF5-induced NOXA protein expression. A, cells were harvested at the indicated times after infection with Ad-hSNF5 and Ad-GFP. Total cell protein (30 μg) were separated on a 4% to 20% SDS-PAGE and probed with either anti-SNF5, anti-β-actin, or anti-NOXA. un; uninfected control. B, cells were transfected with the designated plasmids and selected in neomycin for 2 weeks. The results represent the colony numbers from 3 independent experiments; bars, ±SD.

Figure 4

Recruitment of hSNF5, SWI/SNF complexes, and RNAPII to the NOXA locus and histone modification on NOXA locus after hSNF5 reexpression. A, schematic of the NOXA locus indicating the 1 p53-binding sites and overall gene structure. Primers used in real-time qRT-PCR of ChIP-enriched DNA are named according to their relative distance (bp) to the TSS. B–E, at 24 hours after infection with Ad-hSNF5 and Ad-GFP, protein was extracted for ChIP assays. ChIP assays were conducted using antibodies directed against hSNF5 (B), BRG-1 (C), BAF155 (C), H3K4me3 (C), H3K36me3 (C), RNAPII (D), and p53 (E) on indicated site of NOXA promoter. Values are the mean of duplicate or triplicate; bars, ±SD. F, RNA was extracted at 24 hours after infection with Ad-hSNF5 and Ad-GFP. The mRNA levels were measured for each gene by real-time qRT-PCR and normalized for β-actin expression. Values are the mean of 3 independent experiments; bars, ±SD; un; uninfected control; IP, immunoprecipitation.

Figure 5

Recruitment of hSNF5, SWI/SNF complexes, RNAPII, and p53 to the NOXA locus and histone modification on NOXA locus after hSNF5 reexpression. A, schematic of the p21 locus indicating the 2 p53-binding sites (p53-HABS: high-affinity p53-binding site and p53-LABS: low-affinity p53-binding site) and overall gene structure. Primers used in real-time qRT-PCR of ChIP-enriched DNA are named according to their relative distance (bp) to the TSS. B and C, at 24 hours after infection with Ad-hSNF5 and Ad-GFP, protein was extracted for ChIP assays. ChIP assays were conducted using antibodies directed against hSNF5 (B), BRG-1 (B), BAF155 (B), H3K4me3 (C), H3K36me3 (C), and RNAPII (C) on indicated site of p21 promoter. Values are the mean of duplicate or triplicate; bars, ±SD.