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.