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 c ells, has shortened internucleosome spacings compared with bulk chroma tin or that of the ovalbumin gene, which is inactive. To understand ho w the short (approximately 180-bp) nucleosome repeat arises specifical ly on 13-globin DNA, we have studied chromatin assembly of cloned chic ken beta-globin DNA in a defined in vitro system. With chicken erythro cyte core histones and linker histone H-5 as the only cellular compone nts, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into c hromatin possessing a regular 180 +/- 5-bp repeat, very similar to wha t is observed in erythroid cells. A 2-kb DNA subfragment containing th e beta(A) gene and promoter region, but lacking the downstream interge nic region between the beta(A) and espilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic reg ion 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 1 80-bp nucleosome spacing periodicity observed in erythroid cells is en coded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta(A)-epsilon in tergenic region.