Studies on mat1 Imprinting

用单细胞系谱分析时，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，每四个“孙女”细胞中就有一个经历交配型转换。以前，我们证明这种模式是由于mat1印记，它只以链特异的方式标记一个姐妹染色单体，并与mat1处的位置特异的双链DNA断裂有关。我们现在证明，这个印记是一种链特异性的，在mat1处不稳定的DNA修饰。DNA断裂是一件艺术品，是在DNA纯化过程中从印记中创造出来的。我们还提出并测试了Mat1被着丝粒-远端起源(S)优先复制的模型，因此链特异性印记只发生在滞后链合成过程中。改变复制起点、反转Mat 1或引入复制起点会以预测的方式影响印迹和切换效率。二维凝胶分析证实，mat1是由着丝粒-远端起源(S)优先复制的。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sw1和sw3基因的生化功能。我们发现SWIF1P和SWIF3P通过暂停和终止mat1处的DNA复制来执行印记。我们的工作表明：1)SWIF1P和SWIF3P因子通过在印迹部位暂停复制叉来发挥作用；2)SWIF1P和SWIF3P参与复制的mat1-近端极-终止子(RTS1)的终止。我们进行了基因筛查以确定这些终止因素，并确定了一个等位基因，该等位基因分离了wiw1p的暂停/印记和终止功能。我们的结果表明，SWIF1P和SWIF3P以新的方式促进印记，既通过在mat1暂停复制，又通过在RTS1终止复制。我们还发现，Swi1和Swi3蛋白在体内形成了一个复合体，这两种蛋白都与染色体上的RTS1和mat1复制暂停位点结合。未来的研究将被设计来确定印记的机制。我们已经确定了大量影响印记的mat1突变。对这些mat1突变的分子分析应该有助于我们确定印记的机制。我们一直在寻找另一种系统，在这种系统中，细胞分裂不对称。此外，我们有兴趣将我们的模型应用于到目前为止尚未解释的真核生物的发育表型。由于技术原因，还没有研究确定在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种在其他地方运行这种机制的系统。日本裂殖酵母与庞氏裂殖酵母的蛋白质同源基因在氨基酸水平上只有55%的同源性。尽管进化上存在差异，但mat1处的DNA链特异性表观遗传印记启动了重组事件，这是细胞分化所必需的。因此，S.pombe和S.Japan交配系统提供了前两个例子，其中DNA双螺旋结构的固有链不对称加上DNA复制过程在单个座位上安装的链特定印记构成了细胞不对称分裂的机制。这种机制很容易理解，因为DNA链的不对称为这些单细胞、单倍体生物中姐妹细胞的分化提供了物理基础。裂解酵母的研究已经建立了独特的机制，即链特异的表观遗传标记可以用来赋予接收随后复制的DNA的两个子细胞发育不对称。我们认识到，导致mat1转换现象的细胞不对称分裂机制也可以解释脊椎动物的发育分化导致人体偏侧和大脑半球的不对称发育。然而，为了使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链必须有选择地分离。我们提出了体细胞链特异的表观遗传印迹和选择性姐妹染色单体分离(SSIS)机制，以假设高等真核生物基因组的某些区域利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性或不对称性的机制。SSIS机制已经被提出来解释由于各自的基因突变而导致的身体偏侧发育的变化，以及不同生物中的染色体易位的情况。另外一些例子还包括：由于LRD小鼠的对称内脏器官发育而导致的小鼠胚胎致死率的50%外显率(由RAD51/RAD51基因突变引起的人类先天性手部运动障碍的外显率50%)，以及包含11号染色体易位的家系中50%的精神障碍的外显率。我们认为小鼠的LRD基因和人类的RAD51/RAD51结构对相关染色体进行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，还需要进一步的研究，但每种情况下的发育对称性/不对称性都被认为是在胚胎发育过程中的关键细胞分裂时，恰好两个颗粒细胞实体选择性地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可能已经进化为在不同生物体中完成细胞分化和发育的机制之一。

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.