Studies on mat1 Imprinting

mat1 印迹研究

• 批准号: 8937725
• 负责人: AMAR J KLAR
• 金额: $ 54.59万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8937725
• 关键词: Affect; Alkalies; Alleles; Amino Acids; 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; 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; Organ; Organism; Orthologous Gene; Particulate; Partner in relationship; Pattern; Penetrance; Phenotype; Protein Binding; Proteins; Psychotic Disorders; Replication Origin; Replication-Associated Process; S cerevisiae SWI3 protein; Schizosaccharomyces; Sister; Sister Chromatid; Site; System; Testing; Variant; Visceral; Work; base; daughter cell; design; gene function; genetic pedigree; imprint; in vivo; interest; novel; segregation; termination factor; two-dimensional

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.

当单细胞谱系分析时，pombe Schizosaccharomyces pombe （S. pombe）细胞的开关模式是非随机的。在连续两次不对称细胞分裂后，四分之一的“孙女”细胞经历了交配型转换。先前，我们发现这种模式是由于mat1印迹仅以链特异性的方式标记一个姐妹染色单体，并且与mat1位点特异性的双链DNA断裂有关。我们现在表明，这个印记是一个链特异性的，碱不稳定的DNA修饰在mat1。DNA断裂是一种人工制品，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源复制的模型，因此链特异性印记仅在滞后链合成期间发生。改变复制起始点、反转mat1或引入复制起始点会以可预测的方式影响印迹和转换效率。二维凝胶分析证实，mat1优先由着丝粒远端起源复制。因此，DNA复制机制可能赋予姊妹细胞不同的发育潜能。我们最近的工作已经发现了swi1和swi3基因的生化功能。我们发现swi1p和swi3p通过暂停和终止mat1上的DNA复制来执行印迹。我们的研究表明：1)swi1p和swi3p因子通过在印迹位点暂停复制叉起作用，2)swi1p和swi3p参与了复制的mat1-近端极性终止（RTS1）的终止。我们进行了遗传筛选来鉴定这些终止因子，并鉴定了一个分离swi1p暂停/印记和终止功能的等位基因。我们的研究结果表明，swi1p和swi3p通过暂停mat1位点的复制和终止RTS1位点的复制，以新颖的方式促进印迹。我们还发现Swi1和Swi3蛋白在体内形成复合体，并且这两种蛋白都与染色体上的RTS1和mat1复制暂停位点结合。未来的研究将旨在确定印迹的机制。我们已经发现了大量影响印迹的mat1突变。对这些mat1突变的分子分析将有助于我们确定印迹的机制。我们一直在寻找另一种系统，其中这种不对称细胞分裂机制起作用。此外，我们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，目前还没有研究确定在任何多细胞生物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种系统，这种机制可以在其他地方运作。裂糖酵母菌（Schizosaccharomyces japonicus）裂变酵母菌与已被充分研究的S. pombe菌种高度分化；它们的蛋白质同源物在氨基酸水平上只有55%相同。尽管存在进化差异，但mat1上的DNA链特异性表观遗传印记启动了细胞分化所需的重组事件。因此，pombe和S. japonicus的交配系统提供了最初的两个例子，其中DNA双螺旋结构固有的链不对称加上DNA复制过程在单个位点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链的不对称为这些单细胞单倍体生物的姐妹细胞分化提供了物理基础。裂变酵母的研究已经建立了独特的机制，即链特异性表观遗传标记可以用来赋予两个子细胞发育不对称，这些子细胞随后接受复制的DNA。我们认识到，导致mat1开关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和大脑半球不对称发育的脊椎动物发育分化。然而，为了使这种表观遗传机制在二倍体中发挥作用，来自两条同源染色体的标记DNA链必须被选择性地分离。我们提出体细胞链特异性表观遗传印迹和选择性姐妹染色单体分离（SSIS）机制，假设高等真核生物基因组的某些区域使用表观遗传片段的链标记，然后进行协调的链/染色单体分离，作为建立发育对称或不对称的机制。SSIS机制已被用于解释不同生物体中由于各自的基因突变和染色体易位而导致的身体偏侧发育的变化。在lrd小鼠突变体中，由于内脏器官发育对称而导致的小鼠胚胎致命性的外显率为50%，在人类中，由于rad51/ rad51构成而导致的先天性镜像手运动障碍的外显率为50%，在含有11号染色体易位的家族中，精神病的外显率为50%，这些都是其他这样的例子。我们认为小鼠的LRD基因和人类的rad51/ rad51构成对相关染色体进行了选择性染色单体分离。尽管所有这些系统的机制细节尚不清楚，需要进一步的研究，但每种情况下的发育对称/不对称都是胚胎发生过程中两个颗粒细胞实体在关键细胞分裂时选择性分离到子细胞的结果。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可能是多种生物完成细胞分化和发育的机制之一。