The mammalian X chromosome constitutes a vital biological and clinical model for control of genomic function through formation of facultative heterochromatin, a phenomenon central to normal development and abrogated in cancer. The imprinted XIST gene is required for X inactivation but does not encode a protein. This project investigates the comprehensive hypothesis that sequence-specific interactions between XIST RNA and the chromosome trigger a cascade of chromatin remodeling events that, in the appropriate cellular context, transform an active X chromosome into a highly condensed, transcriptionally inert Barr body. The investigators have shown that an accumulation of stable XIST transcripts structurally associates with the X chromosome, essentially """"""""painting"""""""" the chromosome in cis. Other evidence supports that XIST RNA can induce initiation of chromatin inactivation in the normal developmental context. A central issue now becomes: how does painting by XIST transcripts lead to the sweeping condensation and repression of a whole chromosome? Here key questions will be addressed concerning the interrelationship of biochemical and structural changes involved, and the role of XIST in particular, when expressed either in its native context, in rearranged chromosomes, or as an autosomal transgene induced outside normal developmental context. The investigators' innovative analytical approach which couples precise molecular and biochemical information directly in the context of chromosome structure is important for revealing the impact that XIST RNA has on the chromosome.
Each aim addresses several specific hypotheses as part of each broader hypothesis, which are as follows: 1) That XIST RNA does not itself directly repress transcription, but in developmentally competent cells triggers subsequent changes required for chromosome silencing. 2) That XIST RNA has compromised affinity for autosomal chromatin which results in incomplete and unstable autosomal inactivation. 3) That XIST RNA's physical interaction with the chromosome depends upon specific sequences both in chromosomal DNA and in the XIST transcript. 4) That DNA within an interphase chromosome territory exhibits higher-order organization, which differs on Xi and Xa, and may be medicated directly by XIST RNA. Among the novel studies proposed is a collaborative bioinformatics search for genomic sequence motifs with high affinity for XIST, and the production of an inducible transgene system in which XIST expression can be easily manipulated to define its precise role in initiating inactivation.
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