The chromatin loop or domain model suggests that loops have structural roles in the organization of chromosomes and functional roles in determining the units of eukaryotic gene expression and their functional states. Major questions relate to: i) how the packaging of loops determine the different gene states of a particular cell type i.e. permanently repressed, potentially active and active states; ii) the effects of chromatin variables on DNA topology, nucleosome and chromatin structures and on transcription in constrained loops and iii) the location of the transcriptional machinery relative to chromatin loops and nuclear matices. To address questions i and ii it is essential to use a well-defined system in which nucleosomes are precisely located on known DNA sequences and are correctly spaced. To determine the functions of chromatin variables associated with active chromatin there should be some depth of understanding of transcriptional control of the system. At this time the gene best suited for studies of these broad questions of gene control through substrate availability to transcription factors is the 5S rRNA gene. The 5S rRNA gene with its DNA flanking sequences contains a precise internal TFIIIA binding site and a precise nucleosome core positioning sequence. Simpson's laboratory has engineered plasmids that contain tandem repeats (3 to 50+) of the cloned Lytechinus variegatus 5S rRNA gene and 5' flanking DNA sequences of lengths 172 bp to 207 bp. A closed loop of 50 repeats would provide a model for a chromatin loop of about 10 kbp which is within the rang of loop sizes observed in vivo. We have developed methods to isolate the different states of acetylated and ubiquitinated histones and methods are available to isolate very lysine rich histones and HMG proteins. In preliminary studies we find that the transcription factor TFIIIA binds to acetylated but not to control 5S rRNA gene nucleosomes. In closed circles containing tandemly repeated 5S rRNA genes we now have initial evidence to indicate that the linking number of acetylated nucleosomes is smaller than that of control nucleosomes. This implies that histone acetylation can release nucleosome constrained DNA supercoils to increase the free negative supercoiling within the loop i.e. it may behave as a DNA gyrase. It may also be a mechanism for localizing transient DNA supercoiling between unfolding and refolding of 34 nm supercoils in a transcribing loop, which may explain why the bulk of DNA in chromosomes is in a relaxed state. Two differing views of the mechanism of transcription prevail. In the classical model RNA polymerases are mobile and progress along genes during transcription. In the other model RNA polymerases are anchored to the nuclear matrix and transcribe DNA by reeling it through the attached polymerase complex. We have developed a method for the selective labelling of RNA polymerases engaged in transcription and will determine whether labeled active RNA polymerases are localized on the operationally defined chromatin loops or in the nuclear matrix.
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