분열성 효모에서 수명과 감수분열을 조절하는 Homeobox 전사인자 Phx1의 역할

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2012. 8. 노정혜.분열성 효모에서 phx1+ 유전자는 세포 내에 과량으로 존재할 때Cu / Zn-containing superoxide dismutase (CuZn-SOD) 결핍에 의해 유발되는 lysine 영양결핍증을 극복하는 인자로 처음에 분리되었다. Phx1 단백질을 과량 생산하면, lysine 생합성 과정의 첫 번째 단계에 작용하는 효소이며 동시에 산화 스트레스에 취약한 homocitrate synthase의 합성이 증가한다. Phx1 단백질은 아미노산 말단 근방에 DNA에 결합할 수 있는 잘 보존된 homeodomain을 가지고 있어서 전사조절 인자로 여겨진다. 그러나 Phx1 단백질의 기능은 현재까지 잘 알려지지 않았다. Phx1 단백질의 기능을 연구하기 위해, Phx1 단백질의 발현 패턴과 그것의 결손균주 표현형을 살펴보았다. phx1+ 유전자의 전사량은 세포가 정체 생장기에 들어갈 때 상당히 증가하고, 정체 생장기 내내 높게 유지되었다. 질소와 탄소를 제공하는 영양소의 공급을 낮추면 phx1+ 유전자의 발현량은 지수 생장기에도 증가하는데, 이는 세포가 영양 고갈 환경 시에 Phx1 단백질의 기능을 필요로 함을 의미한다. Phx1에 형광 단백질인 GFP를 결합시켜 형광을 관찰한 결과, Phx1 단백질은 핵 안에 존재하고, 정체 생장기와 영양고갈 시기에 그 발현이 더욱 명확해지는 것을 확인하였다. phx1+ 유전자가 결손된 돌연변이는 장기간의 배양 시에 야생형 균주에 비해 생존률이 감소한다. 반면에 Phx1 단백질을 과량 발현해주면 야생형 균주보다 장기간 생존률이 증가한다. 또한 phx1+ 유전자 결손 돌연변이는 여러 종류의 산화 물질, 열 충격, 알코올 처리 등에 민감하였고, 정체 생장기에 야생형 균주보다 활성 산소량이 더 많이 축적되었다. phx1+ 유전자 결손 이배체의 자낭포자 생성률을 측정한 결과, 야생형 균주보다 상당량 감소되었음을 확인하였다. 전사조절 인자인 Phx1의 조절을 받는 유전자들을 알아내기 위해 microarray 분석을 하였다. 정체 생장기까지 키운 야생형과 phx1+ 유전자 결손 균주의 범유전체적인 전사량 분석을 통해, phx1결손 균주에서 97개 유전자의 발현량이 증가하고 99개 유전자의 발현량이 감소함을 알아내었다. 이 유전자들은 탄수화물 대사, 생식 세포 분화, thiamine 합성, 스트레스 반응, 물질 수송 등 다양한 과정에 참여하였다. Phx1은 특히 pyruvate decarboxylase (PDC)를 위한 유전자인 pdc1+, pdc4+ 와 thiamine 합성을 위한 유전자인 nmt1+, nmt2+, bsu1+ 의 발현을 증가시켰다. 이러한 전사조절 결과와 같은 방향으로, phx1 결손 균주에서 pyruvate decarboxylase의 효소 활성도와 합성된 thiamine의 양이 야생형 균주에 비해 적음을 확인하였다. 게다가, pdc1 과 pdc4 결손 균주는 장기간 생존률이 떨어지는 경향을 보였다. 이것은 pyruvate decarboxylase효소가 Phx1의 조절을 받아 장기간 생존을 유지하는 데 필요한 인자임을 의미한다. Phx1 단백질을 활성화시키는 신호를 찾기 위해, 분열성 효모 내에서 노화를 가속화 시키는 Sck2와 cAMP/Pka1 신호 전달 경로와 Phx1의 관계를 탐구하였다. Phx1 단백질은 Sck2나 Pka1에 의해 인산화될 수 있는 모티프를 가지고 있다. sck2+ 와 phx1+ 유전자가 동시에 결손된 균주와 pka1+ 과 phx1+ 유전자가 동시에 결손된 균주를 제작하여 장기간 생존률을 측정한 결과, phx1+ 결손이 sck2+ 나 pka1+ 유전자가 단독 결손되었을 때 나타나는 수명 연장 효과를 단축시킴을 알 수 있었다. 이는 Sck2와 Pka1이 상위단계에서 Phx1을 부정적으로 조절하는 인자임을 의미한다. 분열성 효모에서 스트레스 반응을 관장하는 Sty1 MAP kinase 신호절달 경로의 수명 조절 효과 및 Phx1 단백질과의 관계 또한 탐구하였다. 흥미롭게도 정체생장기에서 Sty1이 결핍되면, phx1+ 유전자의 발현량이 Sty1의 주요한 타겟 단백질인Atf1 전사조절 인자와 상관없이 감소하였다. 또한, Bimolecular fluorescence complementation (BiFC) 분석을 통해 Sty1과 Phx1 단백질이 물리적으로 결합함을 확인할 수 있었다. 이 결과들은 Phx1이 Sty1의 인산화 타겟이 될 수 있음을 시사한다. 위의 내용들을 종합하자면, Phx1은 정체 생장기와 영양 고갈 시에 발현이 증가하는 전사 조절 인자이다. Phx1은 세포가 장기간 생존하고, 산화, 열, 알코올 등의 스트레스에 저항할 수 있게한다. 또한 감수분열 시에 포자 형성 과정에 중요한 역할을 한다. 더 나아가 이러한 Phx1의 활동은 Pka1, Sck2, Sty1이라는 상위 조절자에 의해 조율을 받는다. 그리고 장기간 생존을 위해 Phx1은 세포 내에서 최소한 두 개의 pyruvate decarboxylase 효소의 발현 및 활성을 증가시킨다. Pyruvate decarboxylase 효소가 어떻게 장기간 생존에 기여하는 지는 앞으로 밝혀야할 과제이다.In the fission yeast Schizosaccharomyces pombe, the phx1+ (pombe homeobox) gene was initially isolated as a multi-copy suppressor of lysine auxotrophy caused by depletion of copper/zinc-containing superoxide dismutase (CuZn-SOD). Overproduction of Phx1 increased the synthesis of homocitrate synthase, the first enzyme in lysine biosynthetic pathway, which is labile to oxidative stress. Phx1 has a well conserved DNA-binding domain called homeodomain at the N-terminal region and is predicted to be a transcription factor in S. pombe. However, its role has not been revealed in further detail. To investigate the functions of Phx1, its expression pattern and the phenotype of its null mutant were examined. The amount of phx1+ transcripts sharply increased as cells entered the stationary phase and was maintained at high level throughout the stationary phase. Nutrient shift down to low nitrogen or carbon sources caused phx1+ induction during the exponential phase, suggesting that cells need Phx1 for the maintenance function during starvation. Fluorescence from the chromosomally encoded Phx1-GFP demonstrated that it is localized primarily in the nucleus, and is distinctly visible during the stationary phase and under nutrient starvation. The phx1 null mutant showed decreased viability in long-term culture, whereas overproduction of Phx1 increased the viability. In addition, the ∆phx1 mutant was sensitive to various oxidants, heat shock, and ethanol treatment. It was found that the ∆phx1 mutant accumulated more reactive oxygen species (ROS) than the wild type at the stationary phase. When we examined sporulation of the ∆phx1 / ∆phx1 diploid strain, significant decrease in the formation of meiotic spores was observed. In order to identify target genes regulated by Phx1, microarray analysis was performed. Genome-wide transcriptional profiling of wild-type and ∆phx1 cells grown to the stationary phase revealed that ∆phx1 mutant increased and decreased the expressions of 97 and 99 genes, respectively. These genes were involved in various biological process including carbohydrate metabolism, sexual reproduction, thiamine synthesis, response to stress, and transport. Phx1 particularly increased the expression of genes for pyruvate decarboxylases (PDC) (pdc1+, pdc4+) and thiamine biosynthesis (nmt1+, nmt2+, and bsu1+). In accordance with transcriptional regulation, the enzyme activity of pyruvate decarboxylase and the amount of thiamine were lower in ∆phx1 mutant than in the wild type. Furthermore, both ∆pdc1 and ∆pdc4 mutants showed defects in long-term survival, suggesting that PDC, being under the control of Phx1, is the primary factor that allow long-term survival. In order to find signals that activate Phx1, we examined the effect of Sck2 and cAMP/Pka1 pathways that are known to have pro-aging effects in S. pombe. It has been noted that Phx1 has motifs that can be phosphorylated by Sck2 or Pka1. Estimation of long-term survival for ∆sck2 ∆phx1 and ∆pka1 ∆phx1 mutants indicated that ∆phx1 counteracted the lifespan extension phenotypes of ∆sck2 and ∆pka1. It suggests that Sck2 and Pka1 negatively regulate Phx1 as upstream regulatory factors. Effect of Sty1 MAPK pathway that regulates cellular stress responses has also been examined. Interestingly, phx1+ expression did not increase at the stationary phase in ∆sty1 mutant, being independent of Atf1 which is a major target of Sty1. Bimolecular fluorescence complementation (BiFC) analysis indicated that Phx1 interacts with Sty1. These results indicate that Phx1 could be a phosphorylation target of Sty1 as well. In summary, this work revealed that Phx1 is a transcriptional regulator whose synthesis is elevated during stationary phase and by nutrient starvation in S. pombe. It functions in long-term survival and stress tolerance against oxidation, heat and ethanol treatment, and plays a key role in the formation of meiotic spores from diploid zygotes. In addition, the activity of Phx1 is modulated by Pka1, Sck2 and Sty1 kinases as upstream regulatory factors. Its contribution to long-term survival is exerted primarily through enhancing the expression and activity of at least two pyruvate decarboxylases in the cell. How PDC can serve such a function requires further study.ABSTRACT i CONTENTS iv LIST OF FIGURES ix LIST OF TABLES xi ABBREVIATIONS xii CHAPTER I. INTRODUCTION 1 I.1. Homeodomain Proteins 2 I.2. Discovery of Phx1 2 I.3. Aging in Yeast 6 I.3.1. Chronological aging 6 I.3.2. Replicative aging 6 I.3.3. Signaling pathways of aging 7 I.4. Meiotic Gene Expression in S. pombe 12 I.5. Pyruvate Decarboxylase (PDC) in Yeast 15 I.5.1. TPP-dependent enzyme family 15 I.5.2. Pyruvate decarboxylase in yeast 17 I.6. Thiamine Biosynthesis in Yeast 18 I.7. Biology of Fission Yeast Schizosaccharomyces pombe 21 I.7.1. Life cycle of S. pombe 22 I.7.2. Cell cycle of S. pombe 26 I.7.3. Genomic structure of S. pombe 26 I.7.4. Deletion mutants library of S. pombe 27 CHAPTER II. MATERIALS AND METHODS 28 II.1. Strains and Culture Conditions 29 II.1.1. Bacterial and yeast strains, media and culture conditions 29 II.1.2. Stress treatments 29 II.2. Recombinant DNA Techniques 31 II.2.1. Plasmids and construction 31 II.2.2. DNA manipulation 33 II.3. Transformation of Escherichia coli and Yeast 33 II.4. Cell Extract Preparation and Analytical Methods 33 II.4.1. Protein extraction and measurement 33 II.4.2. Pyruvate decarboxylase activity assay 34 II.5. RNA Preparation and Analytical Methods 34 II.5.1. RNA isolation and purification 34 II.5.2. Northern hybridization 35 II.5.3. cDNA preparation 35 II.5.4. Quantitative RT-PCR 36 II.6. Gene Disruption and Phenotype Detection 36 II.6.1. Construction of the prototrophic ∆phx1 mutants 36 II.6.2. Construction of multiple disruptants 36 II.7. Measurement of Long-Term Survival 38 II.8. Measurement of Oxygen Consumption 38 II.9. Measurement of Intracellular H2O2 Level 38 II.10. DNA Microarray Analysis 39 II.11. Detection of Mating and Sporulation Efficiency 40 II.11.1. Iodine staining 40 II.11.2. Assay for mating efficiency 40 II.11.3. Assay for sporulation efficiency of diploid 40 II.12. Quantification of Thiamine and Its Derivatives 41 II.12.1. Extraction of thiamine 41 II.12.2. Determination of thiamine and thiamine phosphates by HPLC 41 II.13. Bimolecular Fluorescence Complementation (BiFC) Assay 42 CHAPTER III. RESULTS AND DISCUSSION 43 III.1. Roles of Phx1 in Long-Term Survival 44 III.1.1. Expression of the phx1+ gene increases at the stationary phase and by nutrient starvation 44 III.1.2. Phx1 protein is localized primarily in nucleus at stationary phase and by nutrient starvation 44 III.1.3. The phx1+ gene is required for long-term survival during the stationary phase and under nutrient-starved conditions 47 III.2. Roles of Phx1 in Stress Response 52 III.2.1. ∆phx1 mutant is sensitive to oxidative and heat stress 52 III.2.2. ∆phx1 mutant cells accumulate reactive oxygen species (ROS) at stationary phase 53 III.2.3. ∆phx1 mutant cells show low ethanol tolerance 58 III.3. Roles of Phx1 in Meiosis 60 III.3.1. The ∆phx1 / ∆phx1 diploids are defective in sporulation 60 III.4. Genome-Wide Transcriptional Profiling Analysis of ∆phx1 Mutant 63 III.4.1. The genes that are involved in thiamine biosynthetic and metabolic processes 69 III.4.2. The genes which are related with carbohydrate metabolism & mitochondrial function 72 III.4.3. The genes that are involved in sexual development 73 III.4.4. The genes which are associated with stress response and metabolite transport 74 III.5. Roles of Phx1 in Thiamine Biosynthesis 76 III.5.1. Phx1 regulates transcription of the genes involved in thiamine biosynthesis 76 III.5.2. Intracellular thiamine pools are lower in ∆phx1 mutants than in wild-type cells 79 III.6. Roles of Phx1 in Regulating of PDC Genes 84 III.6.1. Phylogeny of the PDC-like enzymes 84 III.6.2. Phx1 regulates transcriptions of pdc genes 89 III.6.3. PDC activity is decreased in ∆phx1 mutant 92 III.6.4. pdc mutants show long-term survival defect 97 III.6.5. Overproduction of Pdc4 protein enhances long-term survival 100 III.7. Upstream Signaling Pathways for Phx1 Regulation 102 III.7.1. Regulation of long-term survival by the protein kinase Sck2 is mediated via Phx1 102 III.7.2. cAMP/Pka1 pathway negatively regulates Phx1 105 III.7.3. Sty1 MAP kinase pathway may be the upstream regulator of Phx1 112 III.8. Phenotype of Phx1-Overproducing Cells 115 III.8.1. Elongation of cell size 115 III.9. Conclusion 118 CHAPTER IV. REFERENCES 125 국문초록 134 감사의 글 137Docto