Assembly and properties of chromatin containing histone H1

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.     

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.     

A 46 base pair enhancer sequence within the locus activating region is required for induced expression of the gamma-globin gene during erythroid differentiation.

The locus activating region (LAR), contained within 30 kb of chromatin flanking the human beta-globin gene cluster, has recently been shown to be essential for high level beta-globin gene expression. To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma-globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNaseI hypersensitive site II (HS II), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression. We also showed that a 46 bp enhancer element within HS II was necessary and sufficient for the increased gamma-globin gene expression observed with hemin induced erythroid maturation of K562 cells. These results localize a distant regulatory element important for activation of globin genes during human erythroid cell maturation.     

Analysis of the chromatin assembled in germinal vesicles of Xenopus oocytes

We have injected circular DNA, labeled with 32P at a single restriction site, into germinal vesicles of Xenopus laevis oocytes in order to study the nucleosome arrangement on the assembled minichromosomes. Two types of genes were used in these studies, the somatic 5 S RNA gene unit of Xenopus borealis and the histone gene unit of Drosophila melanogaster. We find that injections of labeled DNA alone, at 1 ng DNA per oocyte, results in irregularly spaced nucleosomes and partially supercoiled DNA molecules. However, perfectly spaced nucleosomes are assembled and fully supercoiled DNA is recovered if 5 to 20 nanograms of cold vector DNA is coinjected with the labeled DNA. At the optimum chromatin assembly conditions, the nucleosomes are perfectly spaced with a 180 base-pair periodicity, but they are randomly positioned on the DNA. The assembly of a periodic chromatin structure is accompanied by a dramatic enhancement in the expression of the injected 5 S RNA gene.     

Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region.

The beta-globin locus control region (LCR) is a cis regulatory element that is located in the 5' part of the locus and confers high-level erythroid lineage-specific and position-independent expression of the globin genes. The LCR is composed of five DNase I hypersensitive sites (HSs), four of which are formed in erythroid cells. The function of the 5'-most site, HS5, remains unknown. To gain insights into its function, mouse HS5 was cloned and sequenced. Comparison of the HS5 sequences of mouse, human, and galago revealed two extensively conserved regions, designated HS5A and HS5B. DNase I hypersensitivity mapping revealed that two hypersensitive sites are located within the HS5A region (designated HS5A(major) and HS5A(minor)), and two are located within the HS5B region (HS5B(major), HS5B(minor)). The positions of each of these HSs colocalize with either GATA-1 or Ap1/NF-E2 motifs, suggesting that these protein binding sites are implicated in the formation of HS5. Gel retardation assays indicated that the Ap1/NF-E2 motifs identified in murine HS5A and HS5B interact with NF-E2 or similar proteins. Studies of primary murine cells showed that HS5 is formed in all hemopoietic tissues tested (fetal liver, adult thymus, and spleen), indicating that this HS is not erythroid lineage specific. HS5 was detected in murine brain but not in murine kidney or adult liver, suggesting that this site is not ubiquitous. The presence of GATA-1 and NF-E2 motifs (which are common features of the DNase I hypersensitive sites of the LCR) suggests that the HS5 is organized in a manner similar to that of the other HSs. Taken together, our results suggest that HS5 is an inherent component of the beta-globin locus control region.     

Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes

Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin.