We are studying the mechanisms that maintain constitutive heterochromatic regions in a condensed form. For this purpose we are using a well characterized 16 kb heterochromatin segment located upstream of the chicken beta globin locus. In other published studies we have measured the hydrodynamic properties of this fragment, which is typical of a large proportion of vertebrate genomes, and which has the potential if unregulated to silence adjacent genes in a manner deleterious to cellular function. We found that maintenance of the condensed structure is coupled to low level transcription in the region, and that elevation of levels of histone acetylation by use of histone deacetylase inhibitors markedly increases transcription. Now we have found that the condensed structure is also disrupted in part when histone acetylation increases. We have also found that similar changes can be observed when the enzymes dicer or argonaute2 are depleted: Levels of transcription rise across the domain, histone acetylation increases, and histone H3 K9 methylation decreases, all consistent with opening of the chromatin domain, and the entire region becomes more accessible to attack by the restriction enzyme MspI. Dicer knockdown also results in the disappearance of small interfering RNA molecules derived from the hetercohromatic region. Our results show that mechanisms of maintenance of constitutive heterochromatin in vertebrates may use some of the same mechanisms used by fission yeast, and they show how such domains may be established and maintained in vertebrates. We have now extended our studies to a genome-wide survey of the distribution of the protein Ago2 at repetitive sequences. High concentrations of Ago2 are found at repeat sequences. We are now completing our analysis of these regions.
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