CHROMATIN STRUCTURE AND FUNCTION

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, which is capable of blocking enhancer-promoter interaction when it lies between them. We had earlier narrowed down this activity to a small DNA fragment within the 1.2 kb sequence. We also purified the protein responsible for this activity and identified it as CTCF, which is a known factor ubiquitous in vertebrates, with both stimulatory and inhibitory effects on expression of a variety of genes, and we showed that CTCF sites are present in other vertebrate gene loci, including the 3' end of the chicken beta globin locus. We were also led to investigate a potential insulator activity in the neighborhood of the Igf2 (Insulin-like growth factor 2)/H19 locus in mouse and human. This is an imprinted locus: In the maternally transmitted allele Igf2 is silent and H19 is expressed. The reverse is true in the paternally transmitted allele. A region between the two genes is methylated on the paternal allele only, and it had been suggested that the insulator was within this region. We have now shown that this region contains multiple binding sites for CTCF (four in mouse, seven in human). They serve as very strong enhancer blocking elements, preventing a downstream enhancer from activating Igf2 in the maternal allele. Deletion of these sites abolishes insulation. We also have shown that methylation of the sites prevents CTCF binding. Thus the selective activation of Igf2 on the paternal allele is explained by inactivation of the insulator, which allows the downstream enhancer to turn on this gene. Our results also show that insulator activity can be modulated during development by site methylation. This greatly widens the potential range of action of insulator elements. In other studies we have examined the pattern of chromatin structural changes over the globin locus and its neighborhood during development. We had shown earlier that an erythroid specific folate receptor (FR) gene lies upstream of the chicken beta-globin locus, separated from it by 16 kilobases of condensed non-expressed chromatin. Making use of high resolution immunoprecipitation methods, we have shown that there are major changes in histone acetylation levels over these genes in cells corresponding to various developmental stages, but that the condensed chromatin domain and certain regulatory elements maintain their low and high acetylation levels (respectively) throughout development.