腫瘍抑制遺伝子SMARCB1を欠損した異常なSWI/SNF複合体による遺伝子発現制御の仕組みを解析するため, SMARCB1欠損がん細胞株にSMARCB1を強制発現させて, 増殖能の変化, RNA-seq法を用いて遺伝子発現の変化, ChIP-seq法を用いてSWI/SNF複合体や各ヒストンマークのゲノム上の結合部位や占拠率の変化を解析した。
多くのSMARCB1欠損細胞株でSMARCB1の強制発現により細胞増殖が著しく抑制された。SWI/SNF複合体のサブユニットにはDNA結合部位, ヒストン結合部位が存在するため, 塩化ナトリウムによるDifferential salt extraction法を用いてSWI/SNF複合体のクロマチンとの相互作用を解析したところ, SMARCB1欠損複合体はSMARCB1を含んだ複合体と比較して, 低濃度の塩化ナトリウムでクロマチンと解離した。SWI/SNF複合体の主要なサブユニットに対する抗体を用いたChIP-seq法では, 解析したすべての細胞株においてSMARCB1の強制発現によりSWI/SNF複合体のゲノム上の占有率が有意に増加した。以上の結果より, SMARCB1の欠損はSWI/SNF複合体のクロマチンとの相互作用を不安定化させ, SWI/SNF複合体のゲノム上の占拠率を低下させることで, 遺伝子発現調節の異常をもたらしていることが示唆された。
In order to elucidate the underlying mechanism and to identify direct genetic targets of SMARCB1-deficient aberrant SWI/SNF complexes, we comprehensively evaluated the effects of SMARCB1 reintroduction in SMARCB1-deficient cancer cell lines at the levels of proliferation, global transcriptional signature (RNA-seq), and genome-wide localization (ChIP-seq). Reintroduced SMARCB1 significantly suppressed the proliferation of nearly all SMARCB1-deficient cancer cell lines. As SWI/SNF chromatin remodeling complexes contain several DNA and histone-binding domains, we sought to determine whether SMARCB1 loss alters the stability of BAF complexes on chromatin. We used NaCl-based differential salt extraction to determine the relative affinity of BAF complex proteins on chromatin in cells in either empty or SMARCB1 conditions. We found that SMARCB1-deficient complexes in SMARCB1-deficient cancer cells dissociate from chromatin at the low NaCl concentrations, whereas subunits dissociate from chromatin at the high NaCl treatment upon SMARCB1 reintroduction. Results were similar in comparing SWI/SNF complex chromatin affinity in wild-type and SMARCB1Δ/Δ HEK293T cells. To examine the effect of SMARCB1 rescue on SWI/SNF complex targeting and gene regulation, we performed chromatin IP of SWI/SNF complexes followed by sequencing (ChIP-seq) using antibodies to core SWI/SNF complex subunits. We observed a gain of genome-wide SWI/SNF complex occupancy upon rescue of SMARCB1 in SMARCB1-deficient cell lines. These results indicate that the primary biophysical consequence of SMARCB1 loss is decreased affinity of SWI/SNF complexes for chromatin, suggesting alterations in their genome-wide chromatin occupancy and regulatory capacity.
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