Protein-binding sites within the 5'' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.     

Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin.

Seven cellular loci with acceptor sites for retroviral integrations have been mapped for the presence of DNase I-hypersensitive sites in chromatin. Integrations in three of these loci, chicken c-erbB, rat c-myc, and a rat locus, dsi-1, had been selected for in retrovirus-induced tumors. Of the remaining four, two, designated dsi-3 and dsi-4, harbored acceptor sites for apparently unselected integrations of Moloney murine leukemia virus in a Moloney murine leukemia virus-induced thymoma, and two, designated C and F, harbored unselected acceptor sites for Moloney murine leukemia virus integrations in a rat fibroblast cell line. Each acceptor site mapped to within 500 base pairs of a DNase I-hypersensitive site. In the analyses of the unselected integrations, six hypersensitive sites were observed in 39 kilobases of DNA. The four acceptor sites in this DNA were localized between 0.05 and 0.43 kilobases of a hypersensitive site. The probability of this close association occurring by chance was calculated to be extremely low. Hypersensitive sites were mapped in cells representing the lineage in which integration had occurred as well as in an unrelated lineage. In six of the seven acceptor loci hypersensitive sites could not be detected in the unrelated lineage. Our results indicate that retroviruses preferentially integrate close to DNase I-hypersensitive sites and that many of these sites are expressed in some but not all cells.     

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.     

Developmental regulation of Drosophila DNA topoisomerase II

Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.     

High-resolution mapping of S1- and DNase I-hypersensitive sites in chromatin.

A new and easy technique for accurately mapping DNase I- and S1 nuclease-hypersensitive sites is described. The technique is a modification of primer extension and S1 nuclease methods conventionally used to map RNA ends.     

Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites.

The chromatin conformation of mouse genome regions containing Moloney murine leukemia proviral intergration sites in two Mov mouse strains and randomly selected integration sites in virus-infected mouse 3T3 fibroblasts was analyzed. All integrations have occurred into chromosomal regions containing several DNase-hypersensitive sites, and invariably the proviral integration sites map within a few hundred base pairs of a DNase-hypersensitive site. The probability that this close association between proviral integration sites and DNase-hypersensitive sites was due to chance was calculated to be extremely low (2 X 10(-4]. Because the proviral integrations analyzed were not selected for an altered phenotype, our results suggest that DNase-hypersensitive regions are preferred targets for retrovirus integration.     

Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain.

The distribution of core histone acetylation across the chicken beta-globin locus has been mapped in 15 day chicken embryo erythrocytes by immunoprecipitation of mononucleosomes with an antibody recognizing acetylated histones, followed by hybridization probing at several points in the locus. A continuum of acetylation was observed, covering both genes and intergenic regions. Using the same probes, the generalized sensitivity to DNase I was mapped by monitoring the disappearance of intact genomic restriction fragments from Southern transfers. Close correspondence between the 33 kb of sensitive chromatin and the extent of acetylation indicates that one role of the modification could be the generation and/or maintenance of the open conformation. The precision of acetylation mapping makes it a possible approach to the definition of chromosomal domain boundaries.