Studies on X chromosome inactivation and the X-linked disease Rett syndrome

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references.(cont.) the RTT phenotype.Deletion of the Xist gene results in skewed X-inactivation. To distinguish primary non-random choice from post-choice selection, we analyzed X-inactivation in early embryonic development in the presence of two different Xist deletions. We found that Xist is an important choice element, and that in the absence of an intact Xist gene, the X chromosome will never be chosen as the active X. To understand the molecular mechanisms that affect choice we analyzed the role of replication timing prior to X-inactivation. The X chromosomes replicated asynchronously before X-inactivation but analysis of cell-lines with skewed X-inactivation showed no preference for one of the two Xist alleles to replicate early, indicating that asynchronous replication timing prior to X-inactivation does not play a role in skewing of X-inactivation. Expression of the Xist is negatively regulated by its antisense gene, Tsix. In order to determine the role of transcription in Tsix function, we modulated Tsix transcription with minimal disturbance of the genomic sequence. Loss of Tsix transcription lead to non-random inactivation of the targeted chromosome, whereas induction of Tsix expression caused the targeted chromosome always to be chosen as the active X. These results for the first time establish a function for antisense transcription in the regulation of Xist expression. The X-linked disease Rett syndrome (RTT), a neurodevelopmental disorder, is caused by mutations in the MECP2 gene. We used a mouse model to test the hypothesis that RTT is exclusively caused by neuronal MeCP2 deficiency. Expression of an Mecp2 transgene in postmitotic neurons resulted in symptoms of severe motor dysfunction. Transgene expression in Mecp2 mutant mice, however, rescuedby Sandra Luikenhuis.Ph.D

The SIRT1 Deacetylase Suppresses Intestinal Tumorigenesis and Colon Cancer Growth

Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD+-dependent deacetylases is proposed to underlie the health benefits of calorie restriction (CR), a diet that broadly suppresses cancer in mammals. Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a β-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates β-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of β-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of β−catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in β−catenin-driven malignancies

The Yeast Saccharomyces cerevisiae Contains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins