Mammalian chromosomes are organized as a series of loops called chromatin domains, which behave as independent torsional units and units of DNA replication. The long-term objective of this research is to test the hypothesis that chromatin domains are the functional units of transcriptional competence in that each independent domain can be assembled either as active or as inactive chromatin, and that active chromatin domains complete replication during the first half of S phase, while inactive chromatin domains are often replicated during the second half.
Specific aims are to test four predictions of this hypothesis: (A) The border between active and inactive chromatin should be marked by a structural domain endpoint. (B) Transgenes should behave autonomously if they occupy a chromatin domain of their own, but should behave in a manner consistent with the chromosomal region into which they have inserted if they do not occupy an exclusive domain. (C) Domain border positions should be independent of transcriptional activity. (D)An active domain should replicate during the first half of S phase, while an inactive domain should replicate later in S phase. The research is crucial to understanding correct tissue-specific and stage-specific patterns of gene expression, of the basis for the alterations in expression that occur in cancer, aging, and genetic disease, and of how one might treat genetic disease states (including certain cancers). Position of chromatin domain borders and replication timing will be determined for six chromosomal regions. Replication assays will be performed on both active and inactive copies of genes. Loci to be studied include four transgenes on the mouse X chromosome (transferrin, metallothionein-vasopressin fusion gene, and two independent insertions of tyrosinase), the Xist (inactive- specific transcript) gene, and the H19 and Igf2 loci which are subject to genomic imprinting. Students will have significant input in the design of questions and experiments, carrying out the experiments, in the interpretation of the results, in reviewing the literature, and in the publication and presentation of results, with the objective of encouraging them to consider careers in science. Students will work with cell lines and transgenic mice, using a variety of modern molecular techniques, including nuclear matrix attachment and topoisomerase cleavage site mapping, nuclease sensitivity assays, polymerase chain reaction (PCR), reverse transcription, Northern and Southern blotting, and pulsed-field gel electrophoresis.
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