Prenatal appearance of a short nucleosomal DNA repeat length in neurons of the guinea pig cerebral cortex

The organization of chromatin in neurons of the cerebral cortex of the guinea pig brain was analyzed by digesting isolated nuclei with micrococcal nuclease. During development, cortical neurons were observed to undergo an alteration in chromatin structure which results in an atypically short nucleosomal DNA repeat length of 164 bp. This change in chromatin organization occurs postnatally in certain mammals but in the guinea pig it takes place prior to birth between days 32 and 44 of fetal development. This suggests that the appearance of the short nucleosomal DNA repeat length in cortical neurons correlates to a particular stage of differentiation of cortical neurons rather than to the event of birth.     

Analysis of Histones Associated with Neuronal and Glial Nuclei Exhibiting Divergent DNA Repeat Lengths

Abstract: Total cerebral hemisphere nuclei purified from adult rabbit brain were subfractionated into neuronal and glial populations. Previous studies have shown that chromatin in neuronal nuclei is organized in an unusual nucleosome conformation compared with glial or kidney nuclei, i.e., a short DNA repeat length is present. We now analyze whether this difference in chromatin organization is associated with an alteration in the histone component of nucleosomes. Total histone isolated by acid/urea-protamine extraction of purified neuronal, glial, and kidney nuclei was analyzed by electrophoresis on SDS-polyacrylamide slab gels. Histone H1 that was selectively extracted from nuclei was also examined. Differences were not observed on SDS gels in the electrophoretic mobilities of histones associated with either the nucleosome core particle (histones H2A, H2B, H3, H4) or the nucleosome linker region (histone H1). Total histone and selectively extracted histone H1 were also analyzed on acid/urea slab gels that resolve histones on the basis of both molecular weight and charge differences. When analyzed in this system, differences with respect to electrophoretic mobility were not detected when comparing either selectively extracted histone H1 or total histone from neuronal and glial nuclei. Quantitative analyses were also performed and neuronal nuclei were found to contain less histone H1 per milligram DNA compared with glial or kidney nuclei. Neuronal nuclei also demonstrated a lower ratio of histone H1/core histone. These results suggest that the pronounced difference in chromatin organization in neuronal compared with glial nuclei, which is reflected by a short DNA repeat length in neurons, appears to be associated with quantitative differences in neuronal histone H1.     

The chromatin repeat length of brain cortex and cerebellar neurons changes concomitant with terminal differentiation.

Chromatin repeat lengths in neuronal, glial, and liver nuclei of the rat were determined by micrococcal nuclease digestion followed by gel electrophoresis. The repeat length of cortex neurons decreased from 200 base pairs (bp) before birth to 170 bp at 14 days and all subsequent stages. Administration of [3H]thymidine to pregnant rats during the period of fetal neurogenesis allowed neurons differing in their time of origin to be labeled individually. This revealed that the shortening of the chromatin repeat length affected only neurons generated early during development, i.e., between gestational days 13/14 and 18/19, whereas neurons continuing to proliferate beyond gestational day 19 and up to birth (day 22) did not undergo shortening of their repeat length. In contrast to the cortex neurons, cerebellar neurons (granule cells) underwent lengthening of the repeat length from 165 bp at fetal and early post-natal stages (up to day 4) to 218 bp after day 30. Thus, in both cortex and cerebellar neurons the changes occurred temporally coincident with major developmental processes. No changes were detected in liver nuclei during the same period. Non-astrocytic glia cells of the adult cortex had 200 bp repeats.     

Developmental changes in DNAse I digestibility and RNA template activity of neuronal nuclei relative to the postnatal appearance of a short DNA repeat length

Neurons of the rat cerebral hemispheres are known to undergo a postnatal shift to a short DNA repeat length. In the present study we report that rat neuronal nuclei are more sensitive to digestion with DNAse I when isolated at a developmental stage after the shift in neuronal DNA repeat length compared to nuclei isolated before the shift. This observation may suggest that a decondensation of neuronal chromatin accompanies the postnatal shift in DNA repeat length. We have also found that neuronal nuclei isolated after the shift to a short DNA repeat length demonstrate an increased ability to synthesize RNA in vitro.Abbreviations used op base pairs - PCA perchloric acid - RSB reticulocyte standard buffer - TCA trichloroacetic acid - PMSF phenylmethylsulfonylfuoride     

大鼠老化过程大脑皮层及小脑神经元染色质构象分析

 本文在前文~[2]的基础上进一步以MCN和DNaseⅠ为探针研究大鼠脑神经元终末分化后不同生理时期染色质构象,结果表明:MCN酶解DNA产物PAGE显示脑老化过程大脑皮层及小脑神经元染色质核小体单体DNA分别保持在176bp和215bp水平,核小体连接DNA长度存在组织差异,但不受老化影响;<2>DNaseⅠ酶解DNA产物PAGE显示各年龄组大脑皮层及小脑神经元染色质DNA存在10bp间隔重复结构和相同的泳动区带分布特征,提示脑老化中染色质具有稳定的B型双螺旋结构和一致的螺线管卷曲形式。染色质DNaseⅠ降解率随年龄增加而降低,提示老化导致活性染色质区域减少,老化过程脑神经元染色质构象改变成为其转录功能减退的结构基础。     

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.     

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.     

Different repeat lengths in rat satellite I DNA containing chromatin and bulk chromatin.

The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.     

Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA.

Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA.     

Analysis of putative high-mobility-group (HMG) proteins in neuronal and glial nuclei from rabbit brain

Putative high-mobility-group (HMG) proteins 1, 2, and 17 were detected in neuronal and glial nuclei isolated from the cerebral hemisphere of rabbit brain. Although divergent chromatin structures are present in these two populations of brain nuclei (i.e., neuronal nuclei exhibit a short DNA repeat length), no differences were apparent in the electrophoretic mobilities of putative HMG proteins 1, 2, and 17 on SDS gels.     

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.     

Higher order chromatin structure determines double-nucleosome periodicity of DNA fragmentation

Double-nucleosome periodicity of DNA fragmentation with DNAse I in the nuclei of cells differing in size of the linker DNA length and lysine-rich histone composition was analyzed by means of nondenaturing agarose gel electrophoresis. DNAse I revealed this type of periodicity in rat thymus and CHO cell nuclei as well as in erythrocyte nuclei. It has been deduced that the so-called nucleodisome structure is also typical of cells possessing a usual DNA repeat length (200 bp or less) and lysine-rich histone H1. Two probably related events are important for establishing a clear double-nucleosome periodicity of DNA fragmentation: the replacement of H1 histone by a specific arginine-rich histone fraction (H5 histone in the case of erythrocyte) and the increase of the linker DNA length. The results are interpreted in terms of supranucleosomal organization of chromatin which may determine the dinucleosome periodicity of DNA fragmentation due to a specific packing of nucleosomes.     

Nucleosome rearrangement in vitro. 1. Two phases of salt-induced nucleosome migration in nuclei

We have investigated the salt- and temperature-induced rearrangement of nucleosomes in both intact and H1-depleted nuclei from human cells. In agreement with previous reports on the rearrangement of nucleosomes in isolated chromatin or chromatin fragments, we observed a decrease in the average nucleosome repeat length following incubation of nuclei at 37 degrees C in elevated salt concentrations. However, this decrease occurred in two distinct phases. First, incubation of H1-depleted nuclei at 37 degrees C for as little as 10 min in low-salt, isotonic buffer (containing 0.025 M KCl) resulted in a shift in the limiting repeat value from approximately 190 to 168 base pairs (bp). A similar shift was observed for intact nuclei incubated at 37 degrees C for 1 h in buffer containing near-physiological salt concentrations (i.e., 0.175 M KCl). This limiting repeat value was maintained in both intact and H1-depleted nuclei up to a salt concentration of 0.45 M KCl in the incubation buffer. Second, at salt concentrations of 0.625 M KCl, a limiting repeat of approximately 146 bp was obtained, and the nuclei had clearly lysed. During the first shift in repeat length, little additional exchange of nuclear proteins occurred compared to nuclei kept on ice in a low-salt buffer. This was the case even though the conditions used to monitor exchange were optimized by using a high DNA to chromatin ratio. On the other hand, a significant increase in the exchange of nuclear proteins, and formation of nucleosomes on the naked DNA, was observed during the shift in repeat length to 146 bp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Higher-order structure of nucleosome oligomers from short-repeat chromatin

Sedimentation measurements and electron microscopy at a series of ionic strengths suggest that chromatin from neurons of the cerebral cortex is able to form condensed structures in vitro that are probably several turns of a solenoid with about six nucleosomes per turn. Since neuronal chromatin has a short nucleosomal repeat (approximately 165 bp) allowing virtually no linker DNA between nucleosomes, and yet forms apparently 'normal' elements of solenoid, the packing of nucleosomes in the solenoid must be highly constrained. This permits only a limited number of possible models, and enables tentative suggestions to be made about the location of the linker DNA in the typical solenoid.     

Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts

The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.     

Aspects of large-scale chromatin structures in mouse liver nuclei can be predicted from the DNA sequence

The large amount of non-coding DNA present in mammalian genomes suggests that some of it may play a structural or functional role. We provide evidence that it is possible to predict computationally, from the DNA sequence, loci in mouse liver nuclei that possess distinctive nucleosome arrays. We tested the hypothesis that a 100 kb region of DNA possessing a strong, in-phase, dinucleosome period oscillation in the motif period-10 non-T, A/T, G, should generate a nucleosome array with a nucleosome repeat that is one-half of the dinucleosome oscillation period value, as computed by Fourier analysis of the sequence. Ten loci with short repeats, that would be readily distinguishable from the pervasive bulk repeat, were predicted computationally and then tested experimentally. We estimated experimentally that less than 20% of the chromatin in mouse liver nuclei has a nucleosome repeat length that is 15 bp, or more, shorter than the bulk repeat value of 195 ± bp. All 10 computational predictions were confirmed experimentally with high statistical significance. Nucleosome repeats as short as 172 ± 5 bp were observed for the first time in mouse liver chromatin. These findings may be useful for identifying distinctive chromatin structures computationally from the DNA sequence.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Developmental changes in the synthesis of nonhistone nuclear proteins relative to the appearance of a short nucleosomal DNA repeat length in cerebral hemisphere neurons

Fluctuations in the pattern of synthesis of nonhistone nuclear proteins were noted in cerebral hemisphere neurons during early postnatal development of the rat. Noteworthy changes included the synthesis of an acidic nuclear protein of relative molecular weight 41,000 (41 K), two chromatin-associated basic proteins (37 K and 38 K) and several high molecular weight chromatin acidic proteins. These changes in the synthesis of nonhistone nuclear proteins occur at a developmental stage when a short nucleosomal DNA repeat length has appeared in cerebral hemisphere neurons.Abbreviations used bp base pairs - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IF isoelectric focusing - PMSF Phenylmethylsulfonylfluoride - SDS sodium dodecyl sulphate