SIGNALS IN CHICKEN BETA-GLOBIN DNA INFLUENCE CHROMATIN ASSEMBLY IN-VITRO

We have confirmed the result that chicken beta-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 beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H-5 as the only cellular components, a cloned 6.2-kb chicken beta-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 beta(A) gene and promoter region, but lacking the downstream intergenic region between the beta(A) and espilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta(A) 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 beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta(A)-epsilon intergenic region.