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Studies on mat1 Imprinting

mat1 印迹研究

基本信息

批准号：
9343609
负责人：
AMAR J KLAR
金额：
$ 36.74万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9343609
关键词：
Affect Alkalies Alleles Amino Acids Binding Proteins Biochemical Biological Assay Cell Differentiation process Cell division Cells Centromere Cerebral hemisphere Chromatids Chromosomal translocation Chromosome Pairing Chromosomes Chromosomes, Human, Pair 11 Complex Constitution DNA DNA Double Strand Break DNA Modification Process DNA Structure DNA biosynthesis DNA purification DNA replication fork Development Developmental Gene Diploidy Disease Distal Embryo Embryonic Development Epigenetic Process Eukaryota Event Family Fission Yeast Future Gel Gene Mutation Genes Genetic Recombination Genetic Screening Genome Goals Hand Handedness Haploid Cells Human Mating Types Modeling Molecular Molecular Analysis Morphologic artifacts Movement Disorders Mus Mutant Strains Mice Mutation Organism Orthologous Gene Particulate Partner in relationship Pattern Penetrance Phenotype Proteins Psychotic Disorders Replication Origin Replication-Associated Process Schizosaccharomyces Sister Sister Chromatid Site System Testing Variant Visceral Work base daughter cell design gene function genetic pedigree imprint in vivo interest novel organ growth segregation termination factor two-dimensional

项目摘要

The pattern of switching of Schizosaccharomyces pombe (S. pombe)cells is nonrandom when assayed by single cell pedigrees. After two consecutive asymmetric cell divisions, one in four "granddaughter" cells undergoes a mating-type switch. Previously, we showed that this pattern is due to mat1 imprinting that marks only one sister chromatid in a strand-specific manner, and is related to a site-specific, double-stranded DNA break at mat1. We now show that this imprint is a strand-specific, alkali-labile DNA modification at mat1. The DNA break is an artifact, created from the imprint during DNA purification. We also proposed and tested the model that mat1 is preferentially replicated by a centromere-distal origin(s), so that the strand-specific imprint occurs only during lagging-strand synthesis. Altering the origin of replication, inverting mat1, or introducing an origin of replication affects the imprinting and switching efficiencies in predicted ways. Two-dimensional gel analysis confirmed that mat1 is preferentially replicated by a centromere-distal origin(s). Thus, the DNA replication machinery may confer different developmental potential to sister cells. Our recent work has discovered the biochemical functions of the swi1 and swi3 genes. We found that swi1p and swi3p perform imprinting by pausing and terminating DNA replication at mat1. Our work shows that: 1) the factors swi1p and swi3p act by pausing the replication fork at the imprinting site, and 2) swi1p and swi3p are involved in termination at the mat1-proximal polar-terminator of replication (RTS1). We performed a genetic screen to identify these termination factors and identified an allele that separated the pausing/imprinting and termination functions of swi1p. Our results suggest that swi1p and swi3p promote imprinting in novel ways, both by pausing replication at mat1 and by terminating replication at RTS1. We also showed that Swi1 and Swi3 proteins form a complex in vivo and that both proteins bind to the RTS1 and the mat1 replication pause sites on the chromosome. Future studies will be designed to define the mechanism of imprinting. We have already identified a large number of mat1 mutations that affect imprinting. Molecular analysis of these mat1 mutations should help us define the mechanism of imprinting. We have been looking for another system where such a mechanism of asymmetric cell division operates. Also we are interested in applying our model to hitherto unexplained phenotypes of development in eukaryotes at large. For technical reasons, no studies have been initiated to determine the existence of such a DNA strand-based mechanism of asymmetric cell division in any multicellular organism. We have been searching for another system where such a mechanism operates elsewhere. The Schizosaccharomyces japonicus fission yeast is highly diverged from the well-studied S. pombe species; their protein orthologs are only 55 percent identical at the amino acid level. Despite evolutionary differences, the DNA strand-specific epigenetic imprint at mat1 initiates the recombination event, which is required for cellular differentiation. Therefore, the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic strand asymmetry of the double-helical structure of DNA plus strand-specific imprint installed by the DNA replication process at a single locus constitutes the mechanism of asymmetric cell division. This mechanism is very easy to comprehend because the DNA strands asymmetry provides the physical basis for the sister cells' differentiation in these single-cell, haploid organisms. The fission yeast studies have established the unique mechanism of that strand-specific epigenetic marking can be used to bestow developmental asymmetry upon the two daughter cells that receive the subsequently replicated DNA. We recognized that the mechanism of asymmetric cell division that gives rise to the phenomenon of mat1 switching could also explain the vertebrate developmental differentiation that gives rise to body laterality and asymmetric brain hemispheres development in humans. However, in order for that epigenetic mechanism to work in diploids the marked DNA strand from the two homologous chromosomes will have to be segregated selectively. We proposed the somatic strand-specific epigenetic imprinting and selective sister chromatid segregation (SSIS) mechanism to postulate that certain regions of the genome in higher eukaryotes use the strand marking by epigenetic moiety to be followed by coordinated strand/chromatid segregation as a mechanism to establish developmental symmetry or asymmetry. The SSIS mechanism has been advanced to explain variations of body laterality development due to respective gene mutations and for a case of chromosomal translocations in diverse organisms. The 50 percent penetrance of mouse embryonic lethality due to symmetric visceral organs development in the lrd mouse mutants), the 50 percent penetrance of congenital mirror hand movements disorder due to rad51/RAD51 constitution in humans, and the 50 percent psychoses disorders penetrance in families containing chromosome 11 translocations are other such examples. We propose that he LRD gene in mouse and the rad51/RAD51 constitution in humans function to perform selective chromatid segregation of the relevant chromosome. Although mechanistic details remain unknown for all these systems and require future research, developmental symmetry/asymmetry is proposed in each case to the result of selective segregation of precisely two particulate cellular entities to daughter cells at a critical cell division during embryogenesis. In each case, these entities are probably coincident with the On state of the developmental gene located on non-sister chromatids of a homologous pair of chromosomes. SSIS has likely evolved as one of the mechanisms for accomplishing cellular differentiation and development in diverse organisms.

当通过单细胞谱系进行分析时，粟酒裂殖酵母（S. pombe）细胞的转换模式是非随机的。在连续两次不对称细胞分裂后，四分之一的“孙女”细胞会经历交配型转换。之前，我们表明这种模式是由于 mat1 印记以链特异性方式仅标记一个姐妹染色单体，并且与 mat1 处的位点特异性双链 DNA 断裂有关。我们现在表明，这种印记是 mat1 上的链特异性、碱不稳定 DNA 修饰。 DNA 断裂是一种人工产物，是在 DNA 纯化过程中由印记产生的。我们还提出并测试了 mat1 优先由着丝粒远端起点复制的模型，因此链特异性印记仅在滞后链合成期间发生。改变复制起点、反转 mat1 或引入复制起点会以预测的方式影响印记和转换效率。二维凝胶分析证实 mat1 优先由着丝粒远端复制。因此，DNA复制机制可能赋予姐妹细胞不同的发育潜力。我们最近的工作发现了 swi1 和 swi3 基因的生化功能。我们发现 swi1p 和 swi3p 通过在 mat1 处暂停和终止 DNA 复制来进行印记。我们的工作表明：1）因子 swi1p 和 swi3p 通过在印记位点暂停复制叉来发挥作用，2）swi1p 和 swi3p 参与 mat1 近端复制极终止子（RTS1）的终止。我们进行了遗传筛选来鉴定这些终止因子，并鉴定了一个将 swi1p 的暂停/印记和终止功能分开的等位基因。我们的结果表明 swi1p 和 swi3p 以新颖的方式促进印记，包括在 mat1 处暂停复制和在 RTS1 处终止复制。我们还表明，Swi1 和 Swi3 蛋白在体内形成复合物，并且这两种蛋白均与染色体上的 RTS1 和 mat1 复制暂停位点结合。未来的研究将旨在定义印记机制。我们已经发现了大量影响印记的 mat1 突变。对这些 mat1 突变的分子分析应该有助于我们定义印记机制。我们一直在寻找另一种能够发挥这种不对称细胞分裂机制的系统。我们也有兴趣将我们的模型应用于迄今为止无法解释的真核生物发育表型。由于技术原因，尚未开展任何研究来确定任何多细胞生物体中是否存在这种基于 DNA 链的不对称细胞分裂机制。我们一直在寻找另一个系统，这样的机制可以在其他地方发挥作用。日本裂殖酵母裂殖酵母与经过充分研究的粟酒裂殖酵母种有很大不同。它们的蛋白质直系同源物在氨基酸水平上只有 55% 相同。尽管存在进化差异，mat1 处的 DNA 链特异性表观遗传印记会启动重组事件，这是细胞分化所需的。因此，S. pombe和S. japonicus交配系统提供了前两个例子，其中DNA双螺旋结构的内在链不对称性加上DNA复制过程在单个基因座上安装的链特异性印记构成了不对称细胞分裂的机制。这种机制很容易理解，因为 DNA 链不对称性为这些单细胞、单倍体生物体中姐妹细胞的分化提供了物理基础。裂殖酵母研究已经建立了链特异性表观遗传标记的独特机制，可用于赋予接收随后复制的 DNA 的两个子细胞发育不对称性。我们认识到，引起 mat1 转换现象的不对称细胞分裂机制也可以解释导致人类身体偏侧性和不对称大脑半球发育的脊椎动物发育分化。然而，为了使表观遗传机制在二倍体中发挥作用，来自两个同源染色体的标记 DNA 链必须选择性地分离。我们提出了体细胞链特异性表观遗传印记和选择性姐妹染色单体分离（SSIS）机制，假设高等真核生物中基因组的某些区域使用表观遗传部分的链标记，然后协调链/染色单体分离作为建立发育对称或不对称的机制。 SSIS 机制已得到改进，可以解释由于各自的基因突变和不同生物体中染色体易位的情况而导致的身体偏侧性发育的变化。其他此类例子包括：由于 lrd 小鼠突变体中对称内脏器官发育导致的小鼠胚胎致死性的 50% 外显率、由于人类 rad51/RAD51 体质导致的先天性镜像手运动障碍的 50% 外显率以及含有 11 号染色体易位的家族中 50% 的精神病外显率。我们提出，小鼠中的LRD基因和人类中的rad51/RAD51构成具有执行相关染色体的选择性染色单体分离的功能。尽管所有这些系统的机制细节仍然未知并且需要未来的研究，但在每种情况下都提出发育对称/不对称是胚胎发生过程中关键细胞分裂时精确地将两个颗粒细胞实体选择性分离为子细胞的结果。在每种情况下，这些实体可能与位于同源染色体对的非姐妹染色单体上的发育基因的 On 状态一致。 SSIS 很可能已发展成为多种生物体中完成细胞分化和发育的机制之一。