A phase relationship associates tRNA structural gene sequences with nucleosome cores

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.     

Chromatin structure and expression of a gene transcribed by RNA polymerase III are independent of H2A.Z deposition

The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by approximately 200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5' end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted approximately 50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol III-transcribed gene under different states of its activity in vivo.     

Energy-dependent chromatin accessibility and nucleosome mobility in a cell-free system.

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile. Nucleosome movements, most likely their sliding, occurred even in the presence of the linker histone H1. The dynamic state of chromatin in the presence of the activity and ATP globally increased the accessibility of nucleosomal DNA to incoming proteins. This unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle which is likely to be fundamental to the mechanism of chromatin remodelling and the binding of regulatory proteins.     

In silico approaches reveal the potential for DNA sequence-dependent histone octamer affinity to influence chromatin structure in vivo

Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.