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The RSF1-ATM-p53 signaling axis in conjunction with ATR or DNA-PK upon DNA damage

  • 김기은 (아주대학교 일반대학원 )

초록/요약

Maintaining genome stability is one of the most important factors for proper neural development, so DNA repair mechanisms are crucial for this organ. DNA repair factor mutations could lead to human genetic diseases with genomic instability. Among these genetic diseases, Ataxia Telangiectasia (A-T) affects the brain particularly the cerebellum and the immune system, however its underlying molecular cue particularly in the nervous system is not fully understood. A-T is caused by the mutated ATM gene. ATM, a serine/threonine kinase, responds immediately to DNA double-strand breaks (DSB) which are the most deleterious of DNA damage. Once DSB occur, ATM is recruited to the DNA damage sites and activated to regulate DNA damage responses (DDR) including apoptosis and cell cycle arrest. ATM has numerous downstream substrates including RSF1 and p53. Remodeling and Spacing Factor 1 (RSF1), which is a member of imitation switch (ISWI) family as ATP-dependent chromatin remodelers. RSF1 is involved in transcription regulation, and chromatin remodeling and spacing, when it is associated with SNF2h. In addition, p53 is one of the major substrates of ATM kinase upon DNA damage, which binds to the promoter of the target genes and regulates multiple layers of responses including apoptosis. It is clear that ATM phosphorylates RSF1 and p53 immediately after DNA damage, yet the signaling of RSF1 to p53 and its physiological significance are not fully understood. Thus, I investigated the connection between RSF1 and ATM leading to p53 activity to maintain genomic stability via two different approaches. First one was in vivo analysis of Rsf1 function in the nervous system, particularly related ATM dependent DDR during brain development and A-T neuropathology using the animal model. I used a Nestin-Cre animal to delete the Rsf1 gene selectively in neuroprogenitor cells during neurogenesis resulting in a complete inactivation of RSF1 in the brain. This sole Rsf1 conditional knockout animal model (Rsf1Nes-Cre) did not show any discernible neurological defects, suggesting that RSF1 is dispensable for brain development and function. Dual inactivation of Rsf1 and Atm (Rsf1Nes-Cre;Atm-/-) resulted in no sign of cerebellar ataxia and no neuropathological defects particularly related to A-T neural phenotypes. Next, I treated these genetically modified animals with DNA damage agents to induce DNA damage exogenously and then examined DDR during neurogenesis. I found that the nervous system of the Rsf1Nes-Cre and Rsf1Nes-Cre;Atm-/- showed the dramatic reduction of apoptosis and DDR against exogenously induced DNA damage, yet the residual apoptosis was still sustained in the proliferating ventricular zone, suggesting the possibility of ATR involvement. This reduction influenced the expression of p53 downstream genes, particularly apoptosis related genes, most likely due to inaccessible chromatin status. To study furthermore, I generated U2OS human cell lines of RSF1 knockout (KO), ATM deficiency and RSF1/ATM double deficiency (dKO) using the CRISPR-Cas9 knockout system so that I could analyze the molecular mechanisms of early DDR events in RSF1 deficiency or RSF1/ATM double deficiency, also with combination of ATR or DNA-PK inhibition. RSF1 or ATM deficient cells were impaired in proliferation rates, and RSF1/ATM dKO showed an additive defect in proliferation. After induction of DNA strand breaks or replication stress, DDR showed ATM, ATR and/or DNA-PK dependency. In replication stress induced by etoposide treatment, the ATR signaling became dominant in ATM KO cells, yet was suppressed in RSF1/ATM dKO cells. Random DNA DSB by treatment of phleomycin induced also ATR dependent signaling in collaboration with DNA-PK signaling as an accessary player in ATM KO cells. In RSF1/ATM dKO cells, this collaboration between ATR and DNA-PK was weakened. In general, p53 was not fully activated in RSF1 and ATM double deficiency upon either DNA strand breaks or replication stress. The ATR signaling could be a main substitute in ATM deficiency, yet could not be fully activated in RSF1 and ATM double deficiency. I speculate that RPA (Replication protein A) defective access to single-stranded DNA (ssDNA) is most likely the reason resulting from compacted chromatin status in RSF1 deficiency, which is the critical step to activate ATR kinase function. Now it needs further investigation to verify this speculation at the molecular level.

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초록/요약

신경 발달 과정 중에는 다양한 DNA 손상이 발생되는데, 이러한 손상을 복구하는 과정인 DDR이 매우 중요하다. DDR의 돌연변이로 인하여 신경발달이 제대로 되지 못하게 되고 이는 인간의 유전적 질병으로 이어질 수가 있다. 이러한 유전적 질병들 중에서, A-T는 소뇌와 면역체계에 영향을 미치게 되는 질병이다. A-T는 ATM 돌연변이에 의해 발생된다. DNA 가닥이 끊어지면, ATM은 손상된 DNA에 recruit되어, 다른 하위신호들을 인산화시켜 세포사멸이나 세포주기를 정지시키기 위해 DDR을 활성화시킨다. ATM은 RSF1과 p53을 포함한 수많은 하위신호인자을 가지고 있다. RSF1은 RSF 복합체의 구성요소 중 하나로서 SNF2h와 함께 크로마틴 리모델링 및 전사 조절을 할 수 있는 단백질로 알려져 있다. 그래서 신경계, 특히 뇌 발달 과정에서 RSF1과 ATM의 연관성을 확인하기 위해 Nestin-Cre 동물을 사용하여 Rsf1 유전자를 선택적으로 제거하였다. Rsf1이 불활성화 되어도 신경발달에 영향을 미치지 않았고, 또한 Rsf1과 Atm의 이중 불활성화는 소뇌 실조증의 징후와 신경병리학적 결함을 초래하지 않았다. 그래서 동물 모델에게 DNA 손상을 주는 약물을 투여한 뒤, DNA손상복구를 확인하였다. Rsf1Nes-Cre와 Rsf1Nes-Cre;Atm-/-동물이 신경계에서 외부자극으로 유도된 DNA 손상에 의해 세포사멸이 감소됨을 확인하였다. 이러한 현상은 RSF1이 크로마틴을 열어주어 세포사멸에 중요한 유전자의 발현을 p53이 전사를 유도하지 못하여 발생되었음을 확인하였다. 다양한 실험을 하기 위하여 CRISPR-Cas9을 이용한 유전자재조합을 통해 U2OS 세포주에 RSF1, ATM 및 RSF1/ATM dKO세포주를 제작하였다. RSF1 또는 ATM 이 없는 세포는 느리게 증식하였고, RSF1/ATM dKO는 이보다 더 세포증식에 심각한 결함을 보였다. ATM과 같은 kinase인 ATR 과 DNA-PK 억제제를 처리한 뒤 DNA 손상복구 신호전달의 초기단계를 확인하였다. DNA 가닥 손상 또는 DNA replication stress를 유도하게 되면, ATM, ATR 및 DNA-PK에 의해 활성화된다. ETP 처리에 의해 유도된 replication stress에서 ATR 신호전달은 ATM KO에서 우세해졌지만 RSF1/ATM dKO 에서는 억제되었다. Phleomycin 처리에 의한 무작위 DNA DSB는 ATM KO 세포에서 DNA-PK 신호와 협력하여 보조적으로 ATR 의존적 신호전달을 유도하였다. RSF1/ATM dKO에서 ATR과 DNA-PK 사이의 이러한 협력은 약화되었다. 일반적으로 p53은 DNA strand break또는 replication stress 시 RSF1/ATM dKO에서 완전히 활성화되지 않았다. ATR 신호는 ATM 결핍일때, 대체적으로 작동될 수 있지만 RSF1/ATM dKO 에서는 완전히 활성화되지 못했다. 이는 RSF1 결핍으로 인해 compacted된 크로마틴 상태로 인하여, ATR kinase 기능을 활성화하기 위한 중요한 단계로 RPA가 ssDNA에 접근하기 어려워진 것이 아닌가 하는 가능성을 추측할 수가 있었다. 이에 대한 분자적 메커니즘을 검증하기 위해서는 아직 더 많은 분석이 필요하다.

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목차

CHAPTER I: Introduction 1
A. DNA damage and genomic instability 2
B. DNA double-strand break repair 2
C. DNA damage response (DDR) 4
D. Ataxia Telangiectasia and related human genetic diseases 6
E. RSF1 and ISWI chromatin remodeling complexes 8
F. Hypothesis and Aims of study 10
CHAPTER II: RSF1 function in response to DNA damage in the nervous system 19
A. Chapter Introduction 20
B. Materials and Methods 23
1. Animals 23
2. Western blot analysis 26
3. Histopathological analysis 27
4. Cloning of mouse/human RSF1 gene and micro-irradiation microscopy 28
5. Image and statistical analyses 29
C. Results 30
1. The Rsf1 conditional knockout animal model is successfully generated for the first time. 30
2. Rsf1 is dispensable for brain development and function. 32
3. Atm or p53 deficiency with Rsf1 inactivation does not affect the brain development and neuronal function. 33
4. Rsf1 is involved in response to exogenously induced DNA damage during neurodevelopment. 36
5. Rsf1 deficiency does not influence DNA break itself induced by drug treatments. 38
6. Rsf1 deficiency prevents expressions of p53 dependent genes involved in apoptosis upon DNA damage. 39
CHAPTER III: The involvement of RSF1 in DNA damage response in collaboration with ATM/ATR/DNA-PK 68
A. Chapter Introduction 69
B. Materials and Methods 73
1. Cell culture 73
2. Generation of Knockout cell-line by CRISPR-Cas9 technique 73
3. Drug treatment for inhibiting ATR or DNA-PK and inducing DNA damage 75
4. Western blot analysis 76
5. Cell proliferation and cell cycle analysis 76
6. MNase assay to estimate chromatin status 78
7. Image and statistical analyses 79
C. Results 80
1. RSF1 knockout, ATM knockout and RSF1/ATM double knockout human cell lines are newly generated. 80
2. All of ATM, ATR and DNA-PK play distinct roles in RSF1 depending on the type of DNA damage. 81
3. RSF1/ATM dKO cells display an intermediate level of cell cycle checkpoint between RSF1 KO and ATM KO cells. 87
4. RSF1 and ATM double deficiency could lead to either relaxation or condensation of chromatin depending on the kind of DNA damage. 89
CHAPTER IV: Discussion and Perspective 108
A. RSF1 is dispensable during brain development, but necessary for the chromatin regulation for p53 dependent apoptotic gene expressions upon DNA damage 109
1. The animal model with Rsf1 deficiency in the nervous system different from Snf2h conditional knockout animal model 109
2. Rsf1 is not involved in spontaneously induced DNA damage during neurogenesis 111
3. Apoptosis defects in RSF1 knockout condition 112
B. PIKKs are differently responded to RSF1 proficiency or deficiency 114
1. Differential regulation of ATM, ATR, and DNA-PK signaling in RSF1 KO condition 114
2. Possible implications of these new findings to the clinical setting 115
CHAPTER V: Reference 122

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