Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

Abstract

We have confirmed the result that chicken j3-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on 13-globin DNA, we have studied chromatin assembly of cloned chicken 13-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken j8-globin DNA fragment assembled into chromatin possessing a regular 180 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the 1A gene and promoter region, but lacking the downstream intergenic region between the fA and £ genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the PA gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the 0-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the fA-e intergenic region.Gene regulation in eukaxyotic cells is accomplished through the interplay between transcription factors and chromatin structure (18), but the mechanisms involved in this collaboration are still poorly understood. A popular idea is that in the absence of an activation event, DNA is passively packaged by a default mechanism into an inactive chromatin structure. Upon activation, a gene and its flanking DNA acquire a more open (DNase I-sensitive) chromatin structure (51). Moreover, activation in some cases appears to entail packaging into a large and stable nuclease-sensitive chromatin domain that exceeds by far the region of DNA undergoing transcription (6, 48). Additionally, active chromatin often contains irregular or altered nucleosome arrays (3,12,37,46,49) or exhibits a loss of nucleosome positioning with respect to DNA (7). It has not been adequately explained, however, how a localized activation event could prevent or alter the presumed default packaging of large stretches of DNA into inactive chromatin or, alternatively, how an active chromatin structure could propagate over a large distance. Thus, the tight binding of activator proteins to their recognition sites on DNA would be expected to generate nucleosome-free DNase-hypersensitive sites in that region, as observed (16), but the concomitant opening of a large adjacent chromatin domain would not be expected, given the known physical chemistry of histone-DNA interactions.It has been suggested that the breaking of cooperative interactions among histone Hi molecules in chroma...

Periodicity of strong nucleosome positioning sites around the chicken adult beta-globin gene may encode regularly spaced chromatin.

“…Core histone octamers assembled onto chicken globin DNA under conditions designed to promote the formation of nucleosome arrays adopt a spacing of '180 bp (21). The positioning signals we have detected may well contribute to this spacing, as 180 bp often separates nearest neighbors (Fig.…”

DNA sequence encodes information for nucleosome array formation 1 1Edited by T. Richmond

“…Similarly, cloned Escherichia coli DNA fragments (six out of six) did not assemble into chromatin with physiological nucleosome arrangements (K. Liu & A. Stein, unpublished observations). In contrast with the latter results, it was found that most of the cloned sequences encompassing eukaryotic genes that were examined did assemble into arrays of highly ordered, physiologically spaced, nucleosomes, such that ten or more multiples of the unit repeats could be detected (Lauderdale & Stein, 1992;Liu et al, 1993Liu et al, , 1995Jeong & Stein, 1994). …”

Rat growth hormone gene introns stimulate nucleosome alignment in vitro and in transgenic mice.

“…Because effects of introns on gene expression are often observed when constructs are used to generate transgenic animals, but not when transfected into cultured cells, we favor the idea that appropriate nucleosome alignment, influenced by the DNA base sequence, plays a role in gene regulation during development (3,5). However, because nucleosome alignment in vitro appears to be directed over the rGH gene (with spreading over the promoter region), but not over the 3' flanking sequences, it is plausible that the nucleosome alignment signals in rGH DNA serve to realign nucleosomes after the disruption incurred by the passage of RNA polymerase (17,18).…”

“…Chromatin was assembled in vitro using chicken erythrocyte core histones and linker histone H5 by procedures previously described (5). It was demonstrated, using construct a, that virtually identical results were obtained when histone H5 was replaced by a histone Hi mixture (subtypes a plus b) from chicken erythrocyte.…”

“…A possible explanation for these findings is that genomic DNA might contain sequence arrangements that facilitate the packaging of some genes into chromatin (2)(3)(4)(5)(6). Thus, unnat-ural sequence arrangements might lead to less well-defined chromatin structures that may be deleterious to either transcription initiation or elongation.…”