X-chromosome inactivation (XCI), essential for female mouse development, is the molecular mechanism that ensures equivalent gene dosage of the X-linked genes between XX females and XY males. XCI is the most dramatic example of long-term, chromosome-wide gene silencing in mammalian cells. XCI initiates in early embryogenesis with the expression of the X-inactive specific transcript (Xist) noncoding RNA, which coats the inactivated X-chromosome (Xi) in cis and facilitates the spreading of silencing factors to the entire Xi. Once established, Xi remains silenced through all subsequent somatic cell divisions. Cytogenetic studies indicate that several epigenetic marks are enriched on Xi, including the Xist non-coding RNA and DNA hypermethylation. Moreover, Xi is also enriched with repressive histone marks including H3K27me3, H3K9me3 and H4K20me. However, the protein factors involved in the maintenance of Xi silencing are largely unknown. By performing a genome-wide shRNA screen, we identified 94 genes that are potentially involved in the maintenance of Xi silencing. Following validation of 46 of the 94 candidate genes, 32 genes were verified to be involved in silencing endogenous genes located on Xi. Gene ontology analysis reveals that most of the genes function in RNA processing, cell cycle regulation, gene transcription and chromatin. These results indicate that Xi silencing is maintained via distinct mechanisms. To test this hypothesis, we will first validate which of the remaining 48 genes are involved in Xi silencing and determine how depletion of each verified candidate affects known mechanisms of Xi silencing. Second, we will determine how Orc2 and Lrwd1, both of which were validated in the screen, impact the maintenance of Xi silencing. Orc2 is a subunit of the Origin-Recognition- Complex (ORC), and Lrwd1 is an ORC binding protein. In addition to DNA replication, ORC has a role in epigenetic silencing. However, it was not previously known that ORC and Lrwd1 had any role in Xi silencing. We expect that these studies will provide insight into how epigenetic silencing of Xi is maintained, help delineate the mechanism by which Orc2 and Lrwd1 function in Xi silencing, as well as lay a solid foundation for future studies on the maintenance of Xi silencing.
X-chromosome inactivation (XCI), the most dramatic example of epigenetic silencing, results in repression of expression of most genes on one of the two X chromosomes (Xi) in XX female mammals. XCI is established during early embryogenesis and is then maintained through subsequent cell divisions. It is known that multiple silencing mechanisms, including non-coding RNA, histone methylation (methylation of histone H3 lysine 9 and 27, histone H4 lysine 20) and DNA methylation, work in synergy to maintain silencing. These silencing mechanisms are also adopted by cancer cells to silence tumor suppressor genes (TSG). The protein factors involved in the maintenance of Xi silencing are largely unknown. In this proposal, we plan to identify and characterize genes involved in maintaining Xi silencing. These studies will not only increase our understanding of how Xi silencing is maintained, a poorly understood and critical cellular process, but also shed light on how epigenetic silencing of tumor suppressor genes is maintained.
|Chan, Kui-Ming; Fang, Dong; Gan, Haiyun et al. (2013) The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes Dev 27:985-90|
|Burgess, Rebecca J; Zhang, Zhiguo (2013) Histone chaperones in nucleosome assembly and human disease. Nat Struct Mol Biol 20:14-22|
|Chan, Kui Ming; Han, Jing; Fang, Dong et al. (2013) A lesson learned from the H3.3K27M mutation found in pediatric glioma: a new approach to the study of the function of histone modifications in vivo? Cell Cycle 12:2546-52|
|Chan, Kui Ming; Zhang, Zhiguo (2012) Leucine-rich repeat and WD repeat-containing protein 1 is recruited to pericentric heterochromatin by trimethylated lysine 9 of histone H3 and maintains heterochromatin silencing. J Biol Chem 287:15024-33|
|Liu, Chao-Pei; Xiong, Chaoyang; Wang, Mingzhu et al. (2012) Structure of the variant histone H3.3-H4 heterodimer in complex with its chaperone DAXX. Nat Struct Mol Biol 19:1287-92|
|Chan, Kui Ming; Zhang, Hui; Malureanu, Liviu et al. (2011) Diverse factors are involved in maintaining X chromosome inactivation. Proc Natl Acad Sci U S A 108:16699-704|