CHROMATIN STRUCTURE IN LIVING CELLS

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Chromatin structure has received increasing attention as a modulator of DNA function in transcription, replication, recombination and repair. Most methods for mapping chromatin require isolation of nuclei raising the possibility of alterations i structure during organelle preparation. As one example, the yeast alpha2 repressor is lost form chromatin during preparation of nuclei. We propose a series of investigations to develop methods for mapping chromatin structure in living cells. We have previously utilized the prokaryotic dam methyltransferase to define features of chromatin which preclude access to the enzyme. Recently, we have used a cytosine methyltransferase, expressed from a controlled promoter, which also modifies GATC. The genomic sequencing method for 5 C has been adapted for positive chemical detection, making quantitative analysis of extended regions possible. We will develop methylation methods using more promiscuous enzymes. The Sss I methyltransferase which modifies CpG sequences is available as a cloned gene. We will clone and express genes for Chlorella virus enzymes which recognize CpC and RpCpY sequences. Together, these methyltransferases will allow mapping chromatin with a resolution of one site about every seven base pairs. DNase I was the first enzyme noted to recognize distinctive features of chromatin structure that correlated with DNA function. We tried in the past to express DNase I in yeast to map chromatin in vivo. These attempts failed, likely due to lethality of nuclease expression from a leaky controlled promoter. We have devised several strategies which should allow expression of the nuclease only when desired and will implement then to obtain yeast strains which allow mapping of nuclease hypersensitive sites in living cells as well as detection of the rotational positioning of nucleosomes. The highest resolution, least sequence-specific technique for mapping chromatin in vitro uses hydroxyl radicals. We propose development of hydroxyl radical mapping for chromatin in cells, using gamma radiation for generation of radicals. The studies will be facilitated by previous characterization of positioned nucleosomes abutting the alpha2 repressor in S. cerevisiae minichromosomes, and of a repressed chromatin domain for the STE6 chromosomal gene. We anticipate extension of the methods developed to parallel studies of the structure of 30 kb of yeast chromosome III in our laboratory. While the methodologic development studies are carried out int he tractable environment of S. cerevisiae, there is not reason that these methods can not be exported to higher eukaryotic cells for study of chromatin during development and in disease states.