Studies on mat1 Imprinting

对粟酒裂殖酵母（Schizoschiomycespombe，S.粟酒裂殖酵母）细胞在通过单细胞谱系测定时是非随机的。在两次连续的不对称细胞分裂后，四分之一的“孙女”细胞经历了交配型转换。以前，我们表明，这种模式是由于马特1印记，标志着只有一个姐妹染色单体在一个特定的方式，并与一个位点特异性，双链DNA断裂马特1。我们现在表明，这个印记是一个链特异性，碱不稳定的DNA修饰在mat 1。DNA断裂是一种人工制品，在DNA纯化过程中由印记产生。我们还提出和测试的模型，马特1优先复制的着丝粒远端的起源（S），使链特异性印记只发生在滞后链合成。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记和转换效率。二维凝胶分析证实，mat 1是优先复制的着丝粒远端的起源（S）。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swi 1和swi 3基因的生化功能。我们发现swi 1 p和swi 3 p通过在mat 1处暂停和终止DNA复制来执行印迹。我们的工作表明：1）因子swi 1 p和swi 3 p通过在印记位点暂停复制叉而起作用，2）swi 1 p和swi 3 p参与在复制的mat 1-近端极性终止子（RTS 1）处的终止。我们进行了遗传筛选，以确定这些终止因子，并确定了一个等位基因，分离的暂停/印记和终止功能的swi 1 p。我们的研究结果表明，swi 1 p和swi 3 p促进印迹在新的方式，无论是通过暂停复制在mat 1和终止复制在RTS 1。我们还表明，Swi 1和Swi 3蛋白在体内形成一个复合物，这两种蛋白质结合到RTS 1和染色体上的mat 1复制暂停位点。未来的研究将被设计来定义印迹的机制。我们已经发现了大量影响印记的mat 1突变。对这些mat 1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个系统，在那里这种不对称的细胞分裂机制运作。此外，我们有兴趣将我们的模型应用到迄今无法解释的真核生物发育表型。由于技术原因，没有研究已经开始确定存在这样的DNA链为基础的机制，不对称细胞分裂在任何多细胞生物。我们一直在寻找另一种制度，使这种机制在其他地方运作。裂殖酵母属的裂殖酵母与已被广泛研究的裂殖酵母属的裂殖酵母有很大的不同。粟酒属物种;它们的蛋白质直向同源物在氨基酸水平上只有55%相同。尽管存在进化差异，但mat 1处的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，S. pombe和S.双链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性加上由DNA复制过程在单个位点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链的不对称性为这些单细胞单倍体生物体中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于赋予发育不对称性的两个子细胞，接受随后复制的DNA。我们认识到，导致mat 1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侧化和大脑半球不对称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起作用，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，以假设高等真核生物基因组的某些区域使用表观遗传部分的链标记，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机制已被先进的解释身体偏侧发展的变化，由于各自的基因突变和染色体易位的情况下，在不同的生物体。其他的例子还有：由于第1代小鼠突变体的内脏器官发育对称，导致小鼠胚胎死亡的概率为50%;由于人类的rad 51/RAD 51结构，导致先天性镜像手运动障碍的概率为50%;以及在含有11号染色体易位的家族中，导致精神病障碍的概率为50%。我们认为，小鼠的LRD基因和人类的rad 51/RAD 51组成功能执行相关染色体的选择性染色单体分离。虽然所有这些系统的机制细节仍然未知，需要进一步的研究，发展的对称性/不对称性，提出在每种情况下，在胚胎发生过程中的关键细胞分裂的子细胞的选择性分离的结果，正是两个颗粒细胞实体。在每一种情况下，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.