Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.     

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes.

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.     

Comparison of the active and inactive chromatin structures of genes transcribed by RNA polymerases I and II

The coding sequences of the yeast 35S rDNA gene and of the yeast galactokinase gene both show clear staphylococcal nuclease nucleosome profiles under conditions in which the gene is inactive (galactokinase) or less active (rDNA). Under conditions of more active expression, the galactokinase gene shows marked smearing in the digestion profiles. The rDNA gene shows a qualitatively similar change in digestion patterns. There is a typical nucleosomal DNase I ladder on the coding sequences of both genes, regardless of the state of activity. In contrast to the coding sequences, the rDNA upstream region chromatin shows a nonnucleosomal profile. The nonnucleosomal character is more pronounced when the gene is more active. On the galactokinase upstream region chromatin, there is a nucleosomal structure, with some minor modifications, when the gene is inactive and a clear nonnucleosomal structure when the gene is expressed.     

Structural state of active and inactive genes during chromatin decondensation

Chromatin structure of globin and ovalbumin genes in chicken erythrocyte nuclei has been investigated by means of the "nuclease criterion" (described earlier). In intact nuclei (i.e. in the presence of 3 mM MgCl2) DNase I cleaves chromatin of both genes generating fragments multiple of a double-nucleosome repeat (2N-periodicity). However, in the case of the globin gene, apart from the 2N-periodicity, fragments were observed that are multiple of 100 b.p. and are characteristic for partially unfolded chromatin. This distinction in nuclease cleavage patterns correlates with a higher sensitivity of the globin gene as compared with the inactive ovalbumin gene. At 0.5-0.7 mM MgCl2 the transition from dinucleosomal fragmentation with DNase I and DNase II to fragmentation via a 100 b.p. interval occurs and the difference in digestibility of both genes is dramatically increased. If chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer DNase Il generates an usual nucleosomal repeat, and in this ionic conditions one may not observe any difference in nuclease sensitivity of the analyzed genes. The data allow to suggest that the high nuclease sensitivity of potentially active genes can be conditioned by more relaxed arrangement of nucleosomes in higher order chromatin structure.     

A structure of potentially active and inactive genes of chicken erythrocyte chromatin upon decondensation.

In the presence of 3 mM MgCl2 DNase I cleavage of bulk, globin and ovalbumin gene chromatin in chicken erythrocyte nuclei generates fragments which are multiples of a double-nucleosome repeat. However, in addition to the dinucleosomal periodicity beta-globin gene chromatin was fragmented into multiples of a 100 b.p. interval which is characteristic for partially unfolded chromatin. This distinction correlates with higher sensitivity of beta-globin domain to DNase I and DNase II as compared to the inactive ovalbumin gene. At 0.7 mM MgCl2 where these DNases fragment bulk chromatin into series of fragments with a 100 b.p. interval, the difference in digestibility of the investigated genes is dramatically decreased. When chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer, DNase II generates a typical nucleosomal repeat, and the differential nuclease sensitivity of the analyzed genes is not observed. The data suggest that higher nuclease sensitivity of potentially active genes is due to irregularities in higher order chromatin structure.     

Chromosomal footprinting of transcriptionally active and inactive oocyte-type 5S RNA genes of Xenopus laevis

The chromatin structure of the Xenopus oocyte-specific 5S rRNA genes was examined at high resolution in immature oocyte and somatic cell chromosomes by DNase I footprinting. On oocyte chromatin, where the genes are active, the cleavage preferences over the entire gene region showed a periodic pattern of sensitivity and were dramatically different from the patterns obtained with deproteinized DNA or somatic cell chromatin. Further, the normal binding site for TFIIIA over the internal promoter region was preferentially sensitive to cleavage, indicating that TFIIIA was not bound in the manner predicted by in vitro experiments. In somatic cell chromatin, the oocyte-type 5S genes displayed a cleavage pattern largely similar to deproteinized DNA suggesting the absence of positioned nucleosomes on these inactive genes, although the presence of uncharacterized repressor complexes could not be ruled out. These data are discussed in terms of potential forms of the chromatin structure and alternative mechanisms of oocyte-type gene activation.     

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.