Histone deacetylation is required for the maturation of newly replicated chromatin

The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined. HeLa cells were labeled with [3H]thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min. Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin. However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I. Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA. In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA. Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo. The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated. Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

Hydrodynamic shearing by VirTis blending conserves nucleosome structure of rat liver chromatin

The effects of VirTis shearing on chromatin subunit structure were investigated by enzymatic digestion, thermal denaturation, and electron microscopy. While initial rates of micrococcal nuclease and DNase I digestion were greater postshearing, limit digest values were similar to those for unsheared chromatin. Fractionated chromatin digestion kinetics varied with sedimentation. Digestion of all chromatins produced monomer and dimer DNA fragment lengths, but only unsheared chromatins exhibited higher order nucleosome oligomer lengths. Mononucleosomes and core particles were resolved in digests of sheared and gradient fractions analyzed by electrophoresis. All chromatins exposed to DNase I showed discrete 10-base pair nicking patterns. The presence of nucleosomes was confirmed by electron microscopy. Electron microscopy and histone content of gradient fractions showed that nucleosome density along the chromatin axis increased in rapidly sedimenting fractions. Thermal denaturation detected no appreciable generation of protein-free DNA fragments as a result of shearing. The results indicate that VirTis blending conserves subunit structure with loss of less than 12–15% of nucleosome structure.     

Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Comparison of nuclease digestion of polyoma virus nucleoprotein complex and mouse chromatin.

We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.     

Assembly kinetics of replicating chromatin: Isolation and characterization of prenucleosomal and nucleosomal DNA

Replicating chromatin is known to be more sensitive to micrococcal nuclease than bulk chromatin. We have used this property and a fractionation procedure based on the specific release of replicating material under mild micrococcal nuclease digestion, in order to analyse both the kinetics of maturation of newly replicated DNA into nucleosomes and the structure of the replicating material. As other authors, we initially observed that repetitive unit of newly replicated chromatin was shorter than that of bulk chromatin, however this result appears to be due to sliding of nucleosomes along the chromatin fibers close to the replicating fork. Replicative chromatin was fractionated and analysed. A prenucleosomal peak was observed and preliminary characterized.     

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.     

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.     

Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons

We have used micrococcal nuclease as a probe of the repeating structure of chromatin in four nuclear populations from three tissues of the rabbit. Neuronal nuclei isolated from the cerebral cortex contain about 160 base pairs of DNA in the chromatin repeat unit, as compared with about 200 base pairs for nonastrocytic glial cell nuclei from the same tissue, neuronal nuclei from the cerebellum and liver nuclei. All four types of nuclei show the same features of nucleosomal organization as other eucaryotic nuclei so far studied: nucleosomes liberated by digestion with micrococcal nuclease give a “core particle” containing 140 base pairs as a metastable intermediate on further digestion and a series of single-strand DNA fragments which are mutiples of 10 bases after digestion with DNAase I. Nuclei from cerebral cortex neurons, which have a short repeat, are distinct from the others in being larger, in having a higher proportion of euchromatin (dispersed chromatin) as judged by microscopy and in being more active in RNA synthesis in vitro.     

Nuclease sensitivity of active chromatin.

The active regions of chicken erythrocyte nuclei were labeled using the standard DNase I directed nick translation reaction. These nuclei were then used to study the characteristics and, in particular, the nuclease sensitivity of active genes. Although DNase I specifically attacks active genes, micrococcal nuclease solubilizes these regions to about the same degree as the total DNA. On the other hand micrococcal nuclease does selectively cut the internucleosomal regions of active genes resulting in the appearance of mononucleosomal fraction which is enriched in active gene DNA. A small percentage of the active chromatin is also released from the nucleus by low speed centrifugation following micrococcal nuclease treatment. The factors which make active genes sensitive to DNase I were shown to reside on individual nucleosomes from these regions. This was established by showing that isolated active mononucleosomes were preferentially sensitive to DNase I digestion. Although the high mobility group proteins are essential for the maintenance of DNase I sensitivity in active regions, these proteins are not necessary for the formation of the conformation which makes these genes preferentially accessible to micrococcal nuclease. The techniques employed in this paper enable one to study the chromatin structure of the entire population of actively expressed genes. Previous studies have elucidated the structure of a few special highly prevalent genes such as ovalbumin and hemoglobin. The results of this paper show that this special conformation is a general feature of all active genes irregardless of the extent of expression.     

Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.     

Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs

DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

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.     

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.     

Maturation of newly replicated chromatin of simian virus 40 and its host cell

The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of [³H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication.