Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2006.Includes bibliographical references.A number of mammalian genes are expressed from only one of two alleles in either an imprinted or random manner. Those belonging to the random class include X-linked genes subject to X inactivation, as well as a number of autosomal genes, including odorant receptors, immunoglobulins, T-cell receptors, interleukins, natural killer-cell receptors, and pheromone receptors. Random asynchronous replication of DNA in S-phase represents an epigenetic mark that often parallels monoallelic expression. All randomly monoallelically expressed genes discovered to date replicate asynchronously in S-phase, though not all of the genes contained within asynchronous domains are monoallelically expressed. The focus of my work has been on understanding this random choice that cells make between two sequence-identical alleles. Using two-color fluorescent in situ hybridization (FISH) analyses, the random asynchronous replication of a large number of human and mouse genes appears to be coordinated at the level of entire chromosomes. This regulatory scheme is reminiscent of random X-chromosome inactivation, the dosage compensation machinery in mammals. We have shown that autosomal coordination responds to trisomy in a fashion similar to X inactivation, with one copy of the trisomic chromosome marked for early replication and the other two rendered late replicating.(cont.) These observations raise the intriguing possibility that the mechanistic underpinnings of X inactivation and autosomal coordination may also be similar. Furthermore, the existence of chromosome-wide epigenetic differentiation between autosomes has evolutionary implications concerning the establishment of X inactivation as the approach to mammalian dosage compensation. A crucial event in X inactivation is the random monoallelic expression of a noncoding RNA, Xist from one of the two X chromosomes. Noncoding RNA transcripts are enticing candidates for regulating chromatin structure within the mammalian nucleus. We have initiated a screen for novel nuclear, noncoding RNA transcripts. Using expression array profiling, we have identified several broadly expressed nuclear enriched transcripts. In addition to Xist, this approach identified two noncoding transcripts, NEATI and NEAT2 that are located near one another on human chromosome 1 I and chromosome 19 of mice. Using a variety of techniques, including RNA FISH and RNA-mediated interference, we have explored the potential regulatory functions of these transcripts.by Alexander Wilson Ensminger.Ph.D
