Developmental changes in chromatin organization in rat cerebral hemisphere neurons and analysis of DNA reassociation kinetics

Previous reports have demonstrated that neuronal nuclei of rabbit, mouse and rat cerebral hemispheres exhibit a short DNA repeat length of 160 bp compared to the more typical repeat size of 200 bp found in glial nuclei and other cell types of higher eukaryotes. In this study we report that the conversion of chromatin to a short DNA repeat length in rat cerebral hemisphere neurons is a gradual process which begins between the first and second day after birth and is complete by 8 days. In these neurons, histone H1 appears to be less accessible to degradation by trypsin in the newborn rat brain compared to the 8 day old rat. This suggests that the developmental shift to a short DNA repeat length may be accompanied by a dispersal or decondensation of neuronal chromatin which results in an increased accessibility of neuronal histone H1 to degradation by trypsin. The increase in nuclear DNA content to 3.5C which has been reported in rat cortical neurons during early postnatal development does not appear to be associated with a selective amplification of a subset of DNA sequences as determined by DNA reassociation kinetics.     

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.     

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.     

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.     

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)     

Histones and DNA increase synchronously in neurons during early postnatal development of the rat forebrain cortex

Summary Cytophotometric measurements reveal that a temporally coordinate accumulation of histones and DNA occurs in rat cortex neurons between the last gestational day and the end of the third postnatal week. A rapid rise of both constituents around birth is followed by a more protracted synthesis. This leads to adult histone and DNA levels of approximately 3.3 arbitrary units as compared with 2 arbitrary units found in neuronal precursor cells at all foetal stages and in reference diploid nuclei from glia and liver. A secondary DNA increase in the fourth postnatal week, previously suspected on the basis of cytofluorometric measurements using the Schifftype stain BAO (bis-[4-aminophenyl]-1,3,4-oxadiazole), is not substantiated by the present work. This derives from the finding that alternative cytophotometric DNA measurements (ultraviolet absorption scanning) and autoradiographs of neuronal nuclei following repeated injections of [³H]thymidine into the lateral ventricles during the relevant period give no evidence of a further DNA increase after the third postnatal week. Neither does the accumulation of histones (measured by sulfaflavine cytofluorometry) continue beyond day 21. This leads us to conclude that DNA and histone syntheses cease at the end of the third week. Electrophoretic analyses of the histones show that the relative histone composition changes only slightly during neuronal development. Apart from an increase in the ratio of the histones H1^o:H1 around birth no developmental alterations in histone composition are detectable.     

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.     

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.     

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.     

Decondensation of mouse sperm chromatin and reassembly into nucleosomes mediated by polyglutamic acid in vitro

The organization of the mammalian sperm nucleus was probed with staphylococcal nuclease. Although isolated nuclei are resistant to cleavage, following reduction and alkylation, 30% of the sperm DNA could be digested and the remaining DNA had a heterodisperse size distribution. By morphological criteria, a model acidic protein, polyglutamic acid was capable of decondensing purified sperm nuclei that had been reduced and alkylated. The maximal extent of nuclease digestion increased to 85–90%. The subsequent addition of purified, exogenous core histones in 0.1 M NaCl partially reversed this vulnerability to nuclease cleavage such that only 55% of the DNA was digested. Furthermore, analysis of the remaining DNA revealed a nucleosome ladder pattern with unit length repeat of 150 bp. These results strongly suggest that polyglutamic acid can mediate not only decondensation of sperm nuclei but also the assembly of sperm chromatin into nucleosomes.     

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     

The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).     

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.     

Sequence organization of the rat genome by electron microscopy.

The size and arrangement of repetitive and inverted repeat (foldback) sequences in rat DNA were studied by visualization of hybrid and heteroduplex structures in the electron microscope. The self-reassociation of repetitive sequence-bearing DNA strands often results in the formation of four-ended "H" structures, whose duplex regions equal the repetitive sequence length and can be measured in the electron microscope. In this way, it was determined that the average size of the class of numerous short repetitive sequences is 0.40 +/- 0.15 kbp. Heteroduplex structures were prepared between long whole DNA single strands and short repeat-sequence-bearing strands. The analysis of these structures confirms that the size of the repetitive sequences in 0.4 kbp on average. Length measurements between adjacent duplexes show that the average spacing between two interspersed repeats is at least 1.5-1.8 kbp. By examining 29.4-kbp single strands after brief renaturation, the size and distribution of foldback sequences were determined. There are 1.9 X 10(5) foldback apirs per rat genome, spaced an average of 9.7 kbp apart according to our measurement. Repetitive, inverted repeat and unique sequences are interspersed with each other in at least half the genome.     

Higher order structure in a short repeat length chromatin

Polynucleosomes from calf brain cortical neurone nuclei have an average repeat length of less than 168 base pairs. The ability of this material to adopt higher order structure has been assessed by various physical techniques. Although containing on average less DNA per nucleosome than is required to form a chromatosome, this short repeat length chromatin folded in an H1 dependent manner to a structure with properties similar to those observed for longer repeat length chromatins such as that of chicken erythrocyte (McGhee, J.D., D.C. Rau, E. Charney, and G. Felsenfeld, 1980, Cell, 22:87-96). These observations are discussed in the context of H1 location in the higher order chromatin fiber.     

Postnatal appearance of a short DNA repeat length in neurons of the cerebral cortex

A short DNA repeat length of 162 base pairs was present in neurons of the adult rabbit cerebral cortex while a 198 base pair repeat was found in cortical glial and kidney cells. The short DNA repeat length in cortical neurons was not evident in fetal or new born brain but it appeared during early postnatal development between 2 $\text{1}\text{2}$ and 3 $\text{1}\text{2}$ days in rabbit and between 4 and 7 days in mouse.