Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast.

The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.     

Tissue-specific and periodic changes in the nuclease sensitivity of the fibroin gene chromatin in the silkworm Bombyx mori

Nuclei of substantial purity were isolated from the middle or posterior silk glands of the silkworm Bombyx mori larvae. Both the fibroin H- and L-chain gene sequences in the isolated nuclei from the posterior silk glands of the fifth instar larvae, where the genes are transcribed actively, are extremely sensitive to the digestion with DNaseI; on the other hand, these sequences in the middle silk gland nuclei from the same larvae, where the genes are not expressed, are markedly resistant to the digestion. The H-chain gene sequences in the posterior silk gland nuclei from the fifth instar larvae are also highly susceptible to the digestion with micrococcal nuclease, HinfI, and HhaI. The digestion products with micrococcal nuclease show a continuous size distribution. The H-chain gene sequences in the middle silk gland nuclei or the posterior silk gland nuclei from the fourth molting stage are cleaved partially into nucleosome dimer to oligomer sizes upon digestion with higher concentrations of micrococcal nuclease, suggesting that the inactive forms of the H-chain gene chromatin are constructed by folding of the chromatin fiber containing a regular array of nucleosomes. Hypersensitive sites to micrococcal nuclease are present near both ends of the second exon, a major body of the fibroin H-chain gene, in both the active and inactive forms of the chromatin. The DNaseI or micrococcal nuclease sensitivity of the H-chain gene chromatin in the posterior silk gland nuclei shows periodical changes corresponding to the intermolt-molt-intermolt cycle.     

Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease

The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.     

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.     

Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core

The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions. This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto. The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II. These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core. The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease. While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme. In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA. This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other. The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.     

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.     

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.     

Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena

The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.