Nuclear matrix proteins bind very tightly to specific regions of the chicken histone H5 gene.

The nuclear matrix is operationally defined as the structure remaining after nuclease-digested nuclei are extracted with high concentrations of salt. The nuclear matrix is thought to have a role in organizing higher order chromatin into loop domains. We determined whether specific regions of the histone H5 gene were very tightly bound to protein of erythrocyte and liver nuclear matrices in vitro. We demonstrate that DNA fragments spanning sequences 5' to the promoter and the 3' enhancer region of the histone H5 gene, but not DNA fragments spanning the promoter, were very tightly bound to protein of nuclear matrices of erythrocytes and liver. The nuclear matrix consists of internal nuclear matrix and nuclear pore-lamina complex. Recently, we demonstrated that histone deacetylase could be used as a marker enzyme of the internal nuclear matrix. We demonstrate that nuclear pore-lamina complex preparations that were depleted of histone deacetylase activity, and thus of internal nuclear matrix, retained the protein that bound very tightly to the beta-globin and histone H5 enhancers. These results provide evidence that specific regions of the histone H5 gene are very tightly bound to nuclear pore-lamina complex protein.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer

Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken α-globin silencer-enhancer elements located 3′ of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken α-globin enhancer plays a central role in its capacity to differentially regulate α-globin gene expression during erythroid differentiation and development.     

Status of the nuclear matrix in mature and embryonic chick erythrocyte nuclei

The adult chicken erythrocyte nucleus was found to lack an internal nuclear matrix: even milder extraction procedures resulted in the production of empty shells of pore complex-lamina together with loose aggregates of core histone. In contrast, rat liver nuclei showed a typical intranuclear salt-resistant skeleton. These results show that an internal matrix is not an obligatory nuclear component, and is not required for the spatial organization of chromatin. 5-day-old embryonic erythrocytes did, however, contain an interchromatinic nuclear matrix, suggesting a correlation between the presence of matrix structures, and nuclear 'activity'.