The chromatin structure of an actively expressed, single copy yeast gene.

When the yeast galactokinase gene is not active (repressed, not expressed, quiescent), there is an exceptionally regular nucleosome array on coding sequence galactokinase chromatin, as shown by both denaturing and non-denaturing gel analysis of staphylococcal nuclease digests. Expression of the gene results in a limited smearing of the nucleosome repeat peaks and an increase in interpeak DNA, appearing as a regular ladder of DNA bands on denaturing gels. On non-denaturing gels the pattern is more complex and molecular weight dependent. These data suggest an increase in intracore particle DNA accessibility, allowing staphylococcal nuclease to digest throughout the nucleosome in expressed chromatin. Comparison to bulk chromatin and to an operationally inactive gene (35S rDNA) show that the alteration is specific to expressed chromatin. In contrast, DNase I shows no differences in the digestion of the gene specific chromatin in expressed or inactive states.     

Sex-specific alterations in chromatin conformation of the brain of aging mouse

Chromatin conformation has been analysed in the brain cortex of adult (24±2 weeks) and old (65±4 weeks) male and female mice. Nuclei purified from different groups of mice were digested with MNase and DNase I for varying time periods (0–90 min), and with endogenous endonucleases for 1 h. MNase and DNase I digestion kinetics showed that the percentage of acid solubility of chromatin was relatively lower in old than adult and in female than male. This was further supported by electrophoretic analysis of nuclease digested DNA fragments. When the nuclei were incubated with only Ca²⁺or mg²⁺, no endonuclease digestion was observed. However, under similar conditions, the liver DNA was cleaved substantially. When divalent cations were added together, they activated endogenous endonucleases and digested the brain chromatin. The activity of Ca²⁺/Mg²⁺-dependent endogenous endonucleases was higher in male than female. Thus the accessibility of chromatin to MNase, DNase I and endogenous endonucleases was higher in male than female, and MNase as well as DNase I were more active in adult than old. Such sex- and age-dependent conformation of chromatin may attribute to differential expression of genes in the mouse brain.     

Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status.

There are now many mammalian examples in which single cell assays of transgene activity have revealed variegated patterns of expression. We have previously reported that transgenes in which globin regulatory elements drive the lacZ reporter gene exhibit variegated expression patterns in mouse erythrocytes, with transgene activity detectable in only a sub-population of circulating erythroid cells. In order to elucidate the molecular mechanism responsible for variegated expression in this system, we have compared the chromatin structure and methylation status of the transgene locus in expressing and non-expressing populations of erythrocytes. We find that there is a difference in the chromatin conformation of the transgene locus between the two states. Relative to active transgenes, transgene loci which have been silenced exhibit a reduced sensitivity to general digestion by DNase I, as well as a failure to establish a transgene-specific DNase I hypersensitive site, suggesting that silenced transgenes are situated within less accessible chromatin structures. Surprisingly, the restrictive chromatin structure observed at silenced transgene loci did not correlate with increased methylation, with transgenes from both active and inactive loci appearing largely unmethylated following analysis with methylation-sensitive restriction enzymes and by sequencing PCR products derived from bisulphite-converted genomic DNA.