XIST GENE AND ITS ROLE IN X-INACTIVATION

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X-inactivation is the cis-limited dosage compensation of one of the pair of chromosomes in female mammals. Despite the effort of many research groups, very little has been learned about its mechanism and the number of genes involved. We have isolated a human gene (XIST) which maps to Xq13 within the smallest deletion interval where the X-inactivation center (XIC) has been mapped. The gene encodes for a number of alternatively spliced transcripts expressed exclusively from the inactive X chromosomes. Consequently its expression in chromosomally normal individuals is female- specific. Recently, we have cloned the XIST mouse homolog (Xist) and found that it maps to the mouse X-inactivation center (X controlling element: Xce) region and that its expression is also female-specific in the mouse. The XIST gene because of its mapping assignment and its unique pattern of expression, is an excellent tool to study X-inactivation. The entire murine Xist gene, including the promotor region, will be characterized and sequenced. Sequence comparison between the human and mouse genes will be performed in order to find regions of homology which may represent conserved functional domains. The timing of expression of XIST will be determined in mouse embryos in order to test whether transcription initiates at the time when X-inactivation occurs. Analysis of the XIST gene and its expression will be performed in individuals with chromosome abnormalities resulting in lack of X-inactivation and in mice bearing mutations of the Xce locus. Functional experiments involving knock-out of the XIST gene will be performed in both cultured cells and in embryonal stem (ES) cells from mouse embryos for the creation of transgenic animals. We also plan to isolate overlapping DNA fragments spanning the entire mapping interval where the X-inactivation center has been mapped in human and mouse. Yeast artificial chromosome (YAC), cosmid and lambda phage genomic clones have already been isolated, using XIST as a probe, from both human and mouse libraries and will be used as starting points for chromosome walking. Our studies on the XIST are on the X-inactivation center region will possibly lead to the identification of the function of this unique gene and to the determination of its role in X-inactivation. The unraveling of the molecular basis of X-inactivation, a phenomenon capable of turning off thousands of genes and of acting specifically on only one of the pair of two homologous chromosomes, will certainly lead to the identification of novel mechanisms for the regulation of gene expression.