Chromatin maintenance and sister chromatid differentiation

描述（由申请人提供）：继亲本DNA模板链复制后，姐妹染色单体预计具有完全相同的DNA序列，除了DNA复制本身引起的突变。在细胞分裂过程中，表观遗传标记如何传递给下一代尚不清楚。有丝分裂前未丢失的表观遗传标记要么随机分布在姐妹染色单体之间，要么分配给特定的姐妹染色单体，例如，取决于亲本DNA模板链是通过前导链DNA复制还是滞后链DNA复制进行复制。或者，姐妹染色单体可以以特定链的方式进行印迹。姐妹染色单体之间的染色质差异可能在功能上无关紧要，或者导致子细胞之间基因表达的“随机”差异。姐妹染色单体之间的表观遗传差异也可能涉及细胞命运调节，如果来自“母亲”中心体的微管更喜欢特定染色体的两个姐妹染色单体中的一个上的着丝点。直到最近才能够可靠地识别和区分姐妹染色单体。然而，我们最近发现DNA模板链序列可以可靠地用于鉴定姐妹染色单体。这些新技术将用于研究姐妹染色单体之间表观遗传差异的可能性及其功能意义。拟议的研究有三个具体目的：1。研究与单细胞遗传的特定姐妹染色单体相关的基因表达。2. 研究含有“沃森”或“克里克”DNA模板链序列的姐妹染色单体上的染色质标记。3. 遵循姐妹染色单体的分离，从特定的染色体到特定的细胞类型，在体外和体内。针对这些互补的特定目标提出的研究将澄清姐妹染色单体之间是否存在染色质差异，如果存在，这种差异是否与子细胞之间基因表达的随机变异和细胞命运决定以及多细胞生物的发育有关。重要的是，拟议的研究将回答一个以前没有解决的问题：哪个姐妹染色单体被给定的子细胞遗传有关系吗？如果答案是肯定的，那么提出的研究和实验方法将为进一步研究一个全新的研究领域铺平道路：染色质复制和姐妹染色单体分化。这类研究的意义很难预测，但范围从鉴定新的治疗靶点和从（诱导）多能干细胞开始的更有效的细胞疗法的发展到更有效的靶向正常和恶性干细胞自我更新的策略。

DESCRIPTION (provided by applicant): Following replication of parental DNA template strands, sister chromatids are expected to have exactly the same DNA sequence except for mutations resulting from DNA replication itself. How epigenetic marks are transmitted to the next cell generation during cell division is less clear. Epigenetic marks that are not lost prior to mitosis are either distributed randomly between sister chromatids or assigned to a specific sister chromatid e.g. depending on whether the parental DNA template strand was replicated by either leading or the lagging strand DNA replication. Alternatively, sister chromatids could be imprinted in a strand-specific manner. Chromatin differences between sister chromatids could be functionally insignificant or lead to "stochastic" differences in the expression of genes between daughter cells. Epigenetic differences between sister chromatids could also be involved in cell fate regulation if microtubules originating from "mother' centrosomes were to prefer kinetochores present on just one of the two sister chromatids of specific chromosomes. Until recently it was not possible to reliably identify and distinguish sister chromatids. However, we recently found that DNA template strand sequences can be reliably used to identify sister chromatids. These novel techniques will be used to study the possibility of epigenetic differences between sister chromatids and their functional implications. The proposed studies have three specific aims: 1. Study gene expression in relation to specific sister chromatids that were inherited in single cells. 2. Study chromatin marks on sorted sister chromatids containing either "Watson" or "Crick" DNA template strand sequences. 3. Follow the segregation of sister chromatids from specific chromosomes into specific cell types in vitro and in vivo. The proposed studies towards these complementary specific aims will clarify whether chromatin differences between sister chromatids exist and if so, whether such differences are relevant to stochastic variation in gene expression between daughter cells and cell fate decisions and the development in multicellular organisms. Importantly, the proposed studies will answer a question which has not been addressed before: does it matter which sister chromatids are inherited by a given daughter cell? If the answer is affirmative, the proposed studies and experimental approaches will pave the way for further studies of an entirely novel field of research: chromatin replication and sister chromatid differentiation. The implications of this type of research are hard to predict but range from the identification of novel targets for therapy and the development of more effective cell therapies starting from (induced) pluripotent stem cells to more effective strategies that target self-renewal in normal and malignant stem cells. PUBLIC HEALTH RELEVANCE: Following DNA replication, every chromosome has two sister chromatids which will each end up in the two daughter cells. The proposed studies will answer a fundamental question that could not be addressed before: does it matter which sister chromatid is inherited by a given daughter cell? If the answer is yes, ideas about how disease is caused in a wide spectrum of disorders ranging from cancer to developmental abnormalities will have to be adjusted.