The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction

It was shown with the use of specific probes that mild micrococcal nuclease digestion releases from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A⁺) RNAs, however larger released fragments include some transcribed sequences, while the nuclease-resistant chromatin is considerably impoverished in coding sites. These evidences and the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5–2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure

Chromosome conformation capture (3C) and derivative experimental procedures are used to estimate the spatial proximity between different genomic elements, thus providing information about the 3D organization of genomic domains and whole genomes within the nucleus. All C-methods are based on the proximity ligation–the preferential ligation of joined DNA fragments obtained upon restriction enzyme digestion of in vivo cross-linked chromatin. Here, using the mouse beta-globin genes in erythroid cells as a model, we estimated the actual frequencies of ligation between the fragments bearing the promoter of the major beta-globin gene and its distant enhancers and showed that the number of ligation products produced does not exceed 1% of all fragments subjected to the ligation. Although this low yield of 3C ligation products may be explained entirely by technical issues, it may as well reflect a low frequency of interaction between DNA regulatory elements in vivo.     

RNA促进小鼠重组染色质白蛋白基因DNaseⅠ消化敏感性

同样的基因在不同的分化细胞中表达不同,基因的选择性表达问题涉及分化和衰老的本质。转录基因对DNaseⅠ(DNA酶Ⅰ)消化敏感,本文研究了RNA对小鼠重组染色质白蛋白基因DNaseⅠ消化敏感性的影响。分离BALB/c小鼠脑细胞核,加入终浓度为2mol/L的NaCl破坏核小体结构,加入不同量、不同来源的RNA,装透析袋,逐渐降低离子强度进行染色质重组。重组染色质中加入DNaseⅠ消化DNA,PCR扩增白蛋白基因的外显子1到外显子2约1200bp区段,PAGE电泳后,用银染色观察不同来源RNA促进DNaseⅠ对白蛋白基因的消化作用。不同组织来源（肝、肺、肾、脑）RNA对小鼠重组染色质中白蛋白基因DNaseⅠ消化敏感性均有促进作用,其中肝和肺RNA促进消化作用较强;酵母tRNA无显著促进消化作用;消化促进作用与RNA剂量有关。RNA能增加DNaseⅠ对白蛋白基因的消化敏感性且有组织（细胞）来源特异性。又委托丹麦Chemical R D 实验室合成2条与白蛋白基因互补的各23核苷酸的RNA,用其进行重组试验。结果表明,重组混合物中含有低至0.2μg/mL的RNA,即可以发挥显著的DNase I消化促进作用。     

The telomeric region is excluded from nucleosomal fragmentation during apoptosis, but the bulk nuclear chromatin is randomly degraded

Characteristic steps during cellular apoptosis are the induction of chromatin condensation and subsequent DNA fragmentation, finally leading to the formation of oligomers of nucleosomes. We have examined the kinetics and local distribution of this nucleosomal fragmentation within different genomic regions. For the induction of apoptosis, HL60 cells were treated with the water-soluble camptothecin derivative topotecan (a topoisomerase I inhibitor). The genomic origin of the fragments was analysed by Southern blot hybridisation of the cleaved DNA. In these experiments we observed similar hybridisation patterns of the fragmented DNA, indicating a random and synchronous cleavage of the nuclear chromatin. However, hybridisation with a telomeric probe revealed that, in contrast to the other analysed genomic regions, the telomeric chromatin was not cleaved into nucleosomal fragments despite our observation that the telomeric DNA in HL60 cells is organised in nucleosomes. We determined just a minor shortening of the telomeric repeats early during apoptosis. These observations suggest that telomeric chromatin is excluded from internucleosomal cleavage during apoptosis.     

Spontaneous release of nucleosome cores from embryoblast chromatin

During in vitro incubation of the rabbit blastocyst, chromatin fragments of the nucleosome series are released spontaneously by cells of the pluripotent embryoblast but not by the developmentally restricted trophoblast. The DNA of these fragments is double stranded and linear, and represents total genomic DNA. The fragments are stable for at least 24 hr and are initially found in the cytoplasm of intact cells. The kinetics of radiolabeling of the DNA component of these fragments suggest that they originate from actively replicating cells, possibly only at the time of mitosis. DNase II-like activity, one of the endonucleases known preferentially to attack spacer DNA between nucleosome cores, is readily detectable in the blastocyst, although the evidence for its involvement in the observed chromatin fragmentation remains circumstantial. These nonrandom chromatin breaks, presumably representing an early event in cell death, occur more frequently under suboptimal conditions of incubation but are still detectable in embryos developing normally. There is no evidence for lysosomal activation in these dying cells. These findings, together with previous reports of identical chromatin fragmentation in other cell systems, suggest that this form of cell death in the embryoblast may be only one possible result of an active process, and that the mechanisms responsible for generating double-strand breaks in internucleosomal DNA may be of more biological significance than DNA degradation alone.     

Nick-forming sequences may be involved in the organization of eukaryotic chromatin into ∼50 kbp loops

Phenomena involving the disassembly of chromosomes to ∼50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is ∼100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.     

Mapping networks of physical interactions between genomic elements using 5C technology

Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.     

Accessibility of the ribosomal genes to micrococcal nuclease in Physarum polycephalum.

In Physarum polycephalum most genes coding for ribosomal RNA are not integrated in chromosomes, but are located in many copies in the nucleolus as plasmid-like palindromic DNA molecules. To find out whether coding sequences of rDNA are organized in a chromatin-like structure similar to that of bulk chromatin, nuclei were treated with micrococcal nuclease and DNA fragments were isolated. From bulk chromatin multimers of a basic unit of 170-180 base pairs were obtained. Nuclease fragmented DNA hybridized with labelled 19-S + 26-S rRNA was found to give the same saturation value as did unfragmented control DNA. No preferential degradation of ribosomal genes to acid soluble products was observed. A more detailed analysis of the nuclease degradation products was carried out with fragments separated by preparative gel electrophoresis. DNA eluted from the gels was hybridized in solution with labelled 19-S + 26-S rRNA. The coding sequences of rRNA were found to be degraded to approximately nucleosome size slightly more quickly than was the DNA of bulk chromatin. However, the distribution of the rDNA fragments on the gels did not coincide with the distribution of the fragments derived from bulk chromatin nucleosomes and their oligomers. The amount of rDNA in the interband regions was about intermediate between that found in the two adjacent bands. These results lead to the conclusion that the ribosomal genes, most of which are presumably active during rapid growth, are protected by proteins, probably histones. However, the ribosomal genes are present in a structure differing in some way from that of bulk chromatin.     

Chromatin organization contributes to non-randomly distributed double-strand breaks after exposure to high-LET radiation

The influence of higher-order chromatin structure on the non-random distribution of DNA double-strand breaks induced by high-LET radiation was investigated. Five different chromatin structures (intact cells, condensed and decondensed chromatin, nucleoids and naked genomic DNA) from GM5758 cells or K562 cells were irradiated with (137)Cs gamma-ray photons and 125 keV/microm nitrogen ions (16-25 MeV/nucleon). DNA was purified with a modified lysis procedure to avoid release of heat-labile sites, and fragment size distributions and double-strand break yields were analyzed by different pulsed-field gel electrophoresis protocols. Whereas double-strand breaks in photon-irradiated cells were randomly distributed, irradiation of intact K562 cells with high-LET nitrogen ions produced an excess of non-randomly distributed DNA fragments 10 kb-1 Mbp in size. Complete removal of proteins eliminated this non-random component. There was a gradual increase in the yield of double-strand breaks for each chromatin decondensation step, and compared to intact cells, the yields for naked DNA (in buffer without scavengers) increased 83 and 25 times after photon and nitrogen-ion irradiation, respectively. The corresponding relative biological effectiveness decreased from 1.6-1.8 for intact cells to 0.49 for the naked DNA. We conclude that the organization of DNA into chromatin fiber and higher-order structures is responsible for the majority of non-randomly distributed double-strand breaks induced by high-LET radiation. However, our data suggest a complex interaction between track structure and chromatin organization over several levels.     

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.     

Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

Chromatin can be analysed by assaying its sensitivity to DNase I or other nucleases in purified nuclei. Usually, this is performed by Southern analysis of genomic DNA extracted from nuclease-treated nuclei, a methodology that requires many cells. Applying restriction fragment length polymorphisms (RFLPs), this methodology has been used for parental allele-specific chromatin studies on imprinted mammalian genes. However, such allelic studies are limited by the availability of suitable RFLPs. We therefore developed an alternative, PCR and single strand conformation polymorphism (SSCP)-based assay with which allelic sensitivity to nucleases can be determined in virtually all localised regions that have nucleotide polymorphisms. We also demonstrate that analysis of DNase I sensitivity can be performed on permeabilised cells. Combining the two approaches, in the imprinted mouse U2af1-rs1 gene we analysed parental allele-specific chromatin conformation in limited numbers of cultured cells. We also applied the PCR-SSCP approach to assay allelic DNA methylation at specific restriction enzyme sites. In summary, we developed an allele-specific assay that should be useful for biochemical and developmental investigation of chromatin, in particular for studies on genomic imprinting and X-chromosome inactivation.     

Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells

We here present a general method to detect alkylation damage in specific genomic regions. Cells are treated with nitrogen mustard or dimethyl sulfate; the DNA is extracted and restricted, and the parental DNA is separated. Strand breaks are created at sites of N-alkylpurines by neutral depurination followed by alkaline hydrolysis. The DNA is then separated on alkaline agarose gels and transferred, and gene fragments are detected after hybridization with specific probes. Using this approach, we have examined damage formation and repair in the active genes dihydrofolate reductase and adenosine phosphoribosyltransferase, in a fragment containing the inactive c-fos gene and in a nontranscribed region downstream from the dihydrofolate reductase gene in Chinese hamster ovary cells. We find variations in the formation of nitrogen mustard adducts in these different regions. Nitrogen mustard adducts are preferentially repaired from the active genes as compared to the inactive gene and the noncoding region. However, we find no preferential damage or repair in these regions of the N7-methylpurines after dimethyl sulfate damage. Thus, there are significant differences in the repair mechanisms for two alkylating agents; this may implicate that there are important differences in the structural alterations in chromatin invoked by these agents. As a comparison to the studies of adduct levels in specific genomic regions, we have examined the overall genome, average adduct formation, and repair by these agents in the hamster cells. We used alkaline sucrose gradient sedimentation, and also a novel approach: quantitation of the DNA smears stained by ethidium bromide in the alkaline gels (used in the gene-selective repair analysis). Both these techniques gave similar data for adduct formation and repair; there was less initial damage formation and repair in the average genome than in specific genomic regions.