Molecular Dissection of the Function of BRG-1 in γ-H2AX induction and DNA Double Strand Break Repair

Abstract

세포의 DNA는 화학물질이나 방사선 등에 의해 이중나선이 절단 될 수 있다. 이러한 이중나선 절단 (DSBs) 은 가장 위험한 종류의 손상이므로, 세포는 세포주기의 정지, 세포 사멸 그리고DNA 손상 치유 과정을 통해 암으로의 전이를 막고 있다. 최근 들어ATP 의존적인 크로마틴 리모델링 복합체인 SWI/SNF 복합체가 종양 억제 유전자로서 기능한다는 증거가 밝혀지고 있다. 특히 SWI/SNF 복합체가 히스톤 단백질인 H2AX의 인산화를 촉진시켜 DNA 손상 치유 과정에 참여한다는 사실이 이미 밝혀져 있었지만 그 자세한 메커니즘은 밝혀지지 않았다. 따라서 본 논문에서는 SWI/SNF 복합체가 어떻게 히스톤의 인산화와 DNA 손상 치유에 관여하는지를 분자생물학적인 관점에서 조사하였다. 먼저 RNA 간섭 기법을 통해 SWI/SNF 복합체가 인산화 된 H2AX에 필수적임을 밝혀내었는데, RNA 간섭으로 SWI/SNF 복합체의 주요 단백질인 BRG-1의 발현을 감소시키면 H2AX의 인산화가 줄어들었다. 한편으로 add-back 기법을 통해 RNA 간섭에 공격받지 않는 BRG-1을 발현시킴으로써 H2AX의 인산화와 DNA 손상 치유에 SWI/SNF 복합체가 중요한 역할을 한다는 것을 다시 한 번 확인하였다. 그 다음 ATPase 변이와 bromodomain 결실 등의 실험기법을 통하여BRG-1의 bromodomain이 H2AX의 인산화와 DNA 손상 치유에 필수적임을 밝혀내었다. 하지만 ATPase 활성화는 DNA 손상 치유에는 중요한 역할을 하였으나, H2AX 인산화를 촉진시키는 데는 필수적이지 않았다. 이러한 결과로 H2AX의 인산화와 히스톤 H3의 아세틸화, SWI/SNF 복합체 그리고 DNA 손상 치유에 이어지는 생물학적인 시스템 모델을 세울 수 있었다. 본 논문은 포유류에서 처음으로, 크로마틴 리모델링 복합체가 H2AX의 인산화와 DNA 손상 치유에 어떻게 작용하는지 그 메커니즘을 분자생물학적인 측면에서 처음으로 밝혀내었다는 점에서 의의가 있다고 할 수 있다.;Recent evidence suggests that the SWI/SNF complexes, the founding members of ATP-dependent chromatin remodeling complex family, function as tumor suppressor. In support of this notion, our laboratory has recectly shown that the SWI/SNF complexes play important roles in DNA double strand break (DSB) repair and the maintenance of genome integrity. Data suggest that the SWI/SNF complexes facilitate DSB repair by promoting the phosphorylation of histone H2AX (gamma―H2AX) via direct binding to the chromatin surrounding the DSB sites. However, the mechanisms underlying these functions of the SWI/SNF complexes are not known. The research of this thesis aimed at understanding how the SWI/SNF complexes facilitate the gamma―H2AX induction and DSB repair at the molecular levels. I started by demonstrating the role of the SWI/SNF complexes in gamma―H2AX and DSB repair by using small interfering RNA (siRNA) approches. I found that downregulation of both BRG-1 and hBrm, the catalytic subunits of the SWI/SNF complexes, compromises gamma―H2AX induction as well as DSB repair. I then generated a expression vector that produces BRG-1 mRNA that is resistant to the action of BRG-1 siRNA, and performed add back experiments. I found that re-expression of BRG-1 in the cells in which BRG-1 was downregulated by siRNA fully rescued the gamma―H2AX induction and DSB repair. These results clearly demonstrated that the SWI/SNF complexes are specifically responsible for both gamma―H2AX induction and DSB repair. I further went on to generate a series of siRNA-resistant mutant constructs, and examined which domains of BRG-1 are responsible for gamma―H2AX induction and DSB repair. I found that either mutation of Lys-798 to Arg on the ATPase domain, known to abrogate the chromatin remodeling activity of the complexes in vitro, or deletion of the bromodomain, known to bind to acetylated histones, did not rescue DSB repair when they were expressed in the BRG-1 knockdowned cells, indicating that both chromatin remodeling activity and the bromodomain are important for DSB repair. Very interestingly, however, the induction of gamma―H2AX following DNA damage was severely compromised by the bromodomain deletion, but not affected at all by the ATPase mutation. These data suggest that gamma―H2AX and DSB repair require distinct activities of the SWI/SNF complexes, and that the SWI/SNF complexes contribute to DSB repair not only by promoting gamma―H2AX but also via other pathways requiring the ATPase activity. Recently, our laboratory observed that the H2AX phosphorylation is required for the acetylation of Lys-14 of histone H3 at the sites of DSB. In addition, a recent work showed that the bromodomain of BRG-1 interacts specifically with acetylated H3 peptides on Lys-14 in vitro. By integrating these results with my findings, I proposed a model in Discussion for the molecular mechanisms for the role of BRG-1 in gamma―H2AX induction and DSB repair.