Chromatin Repeat Research Articles

Topological constraints on the possible structures of the 30 nm chromatin fibre

The packing of mammalian DNA into chromatin plays an important role in cell differentiation and selection of epigenetically marked genes for expression or silencing. The first level of folding, the nucleosome, is evolutionary conserved. It allows transcription, after remodeling and/or histone modifications. The second level, the transcriptionally dormant 30 nm fibre, exhibits species and tissue variations in the chromatin repeat length. Nevertheless, very similar structures of fibres have been observed in all metazoans, and therefore, have to accommodate variable linker lengths with a corresponding change of tilt of the nucleosomes, which is defined by the DNA helical periodicity. So far, none of the models for a regular fibre structure has considered this requirement nor the relationship between repeat length and orientations of nucleosomes in the fibre. Here, we present two regular structural arrangements with negatively tilted consecutive nucleosomes which can compensate for a non-integer number of bp/turn of DNA; one can accommodate a series of structures with discrete repeat lengths differing by 5 bp in the region around 200 bp and the other from around 220 to 250 bp, accommodating repeat lengths differing by 10 to 12 bp.

DNase I digestion reveals alternating asymmetrical protection of the nucleosome by the higher order chromatin structure.

DNase I was used to probe the higher order chromatin structure in whole nuclei. The digestion profiles obtained were the result of single-stranded cuts and were independent of pH, type of divalent ion and chromatin repeat length. Furthermore, the protection from digestion of the DNA at the entry/exit points on the nucleosome was found to be caused not by the H1/H5 histone tails, but by the compact structure that these proteins support. In order to resolve symmetry ambiguities, DNase I digestion fragments over several nucleosome repeat lengths were analysed quantitatively and compared with computer simulations using combinations of the experimentally obtained rate constants (some of which were converted to 0 to simulate steric protection from DNase I digestion). A clear picture of precisely defined, alternating, asymmetrically protected nucleosomes emerged. The linker DNA is inside the fibre, while the nucleosomes are positioned above and below a helical path and/or with alternating orientation towards the dyad axis. The dinucleosomal modulation of the digestion patterns comes from alternate protection of cutting sites inside the nucleosome and not from alternating exposure to the enzyme of the linker DNA.

Influence of chromatin molecular changes on RNA synthesis during embryonic development.

Two aspects of the chromatin repeat length (rl) are discussed: (i) Why is rl longer for slowly dividing cells than in rapidly dividing cells?, and (ii) Why is the temporal evolution of rl a decreasing function of time (t) in mammalian cortical neurons, whereas it is an increasing function of t for granule cells around the time of birth? These questions are discussed in terms of a hypothesis which assumes a correlation between deoxyribonucleic acid (DNA) packaging, transcription, and replication.

Triiodothyronine-induced shortening of chromatin repeat length in neurons cultured in a chemically defined medium.

At the time of terminal differentiation, mammalian cortical neurons undergo a dramatic change in the structural organization of their chromatin: the nucleosomal repeat length shortens from approximately 200 base pairs in fetuses to a value of 165 base pairs after birth. These events occur several days after the end of neuronal proliferation. Previously, we reported that rat cortical neurons cultured in a very selective synthetic medium were not yet programmed to these events at the end of mitotic cycles. Herein, we report that addition of triiodothyronine to neuronal cultures induces a shortening of the chromatin repeat length comparable to the natural one.

Rat CNS neurons are not yet programmed to shorten their chromatin repeat length at the end of fetal neurogenesis

Neurons from rat fetal cerebral hemispheres were grown in a synthetic medium (Maat medium), as previously described, for different periods of time. The repeat length of their chromatin was determined by micrococcal nuclease digestion and compared with that of neurons isolated from postnatal rat brain of corresponding ages. In contrast to the in vivo situation, we found that neurons, dissociated at the 16th gestational day and cultured in vitro, did not undergo the shortening of their chromatin repeat, thus indicating that, at the end of their mitotic cycles, they are not yet programmed to this event.

A low repeat length in oligodendrocyte chromatin.

The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."

T-DNA of a crown gall tumor is organized in nucleosomes

Nucleosomes were isolated from chromatin of suspension cultured cells of Nicotiana tabacum var. White Burley, which were either habituated or transformed by Agrobacterium tumefaciens, strain T37. Chromatin repeat length in both types of tissue was identical and can be estimated to be 195 +/- 10 bp. Using Southern transfer of nucleosomal DNA and hybridization with cloned nick-translated HindIII fragments of pTi C58 we show that the T-DNA originating from the Ti-plasmid of A. tumefaciens is organized in nucleosomes within the chromatin of crown gall tumor cells.

Physical structure of gene-sized chromatin from the protozoan Oxytricha.

Oxytricha nova is a hypotrichous ciliate containing a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Two-dimensional gel electrophoresis shows that macronuclei contain a normal complement of inner histones. However, despite extensive efforts, no classical H1-like protein has been detected. Micrococcal nuclease digestion indicates a nucleosome repeat length of approximately 220 bp for macronuclear chromatin. Thermal denaturation profiles of macronuclear chromatin in 0.2 mM EDTA display four transitions at about 46, 57, 64, and 79 degrees C. The lowest of these shifts to higher temperature as the ionic strength is raised to 3-5 mM Na phosphate. These results are consistent with the absence of H1 and a nucleosome repeat of 220-230 bp. Circular dichroism (CD) results agree with these findings. By contrast, micronuclear chromatin displays a much smaller premelt and a more suppressed DNA CD signal at 285 nm, consistent with a micronuclear chromatin repeat of 165-185 bp as determined by micrococcal nuclease digestion.

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.

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.

Chromatin repeat length correlates with phenotypic expression in hepatoma cells, their dedifferentiated variants, and somatic hybrids.

Chromatin repeat length is known to differ among the tissues of organisms. We have examined the repeat length of somatic cells in culture and have found an unexpected correlation between this parameter and the state of differentiation of the cells. Three sequentially derived subclones of a rat hepatoma, similar in phenotype, present a constant repeat length of 189 (+/- 1) base pairs. Three independent variant clones do not express hepatic functions and each has a different repeat length: 184 base pairs, 188 base pairs, and 192 base pairs. Somatic hybrids between the two types of cells in which liver functions are extinguished have a repeat length close or identical to that of the nonexpressing parent. In the case of one hybrid clone that reexpresses the liver phenotype and has undergone some chromosome loss, the 189-base-pair repeat length characteristic of the well-differentiated cells is reestablished. Finally, an amelanotic variant clone derived from a pigmented hamster melanoma clone also shows a signficantly altered repeat length. No correlations have been observed between repeat length and either generation time or chromosome number. Maintenance of the expression of differentiation therefore appears to be associated with a constant repeat length, and loss of the former with a modification in repeat length, suggesting that chromatin packaging is intimately involved in the regulation of gene expression.

Chromatin repeat length in somatic hybrids.

In order to study the mechanisms by which a characteristic repeat length is inherited in somatic cells, it was necessary to develop a method for determining repeat length with a precision of 1 to 2 base pairs. Hybrid clones between parental cell lines differing in repeat length by 6 base pairs were isolated. The four independent hybrid clones characterized had repeat lengths intermediate between those of the parental lines; however, it could be demonstrated that these repeat lengths are unique values and do not arise from a double distribution of the parental repeat lengths. It therefore is concluded that repeat length in somatic cells is determined by a common pool of diffusible substances.

The chromatin repeat length of cortical neurons shortens during early postnatal development

The chromatin repeat length of cortical neurons shortens during early postnatal development

Analysis of chromatin repeat units in logarithmically and stationary growing cells of paramecium aurelia and tetrahymena pyriformis

Abstract We have used micrococcal nuclease as a probe of the repeating structure of chromatin isolated from the macronuclei of logarithmically and stationary grown Paramecium aurelia and Tetrahymena pyriformis . For both these lower eukaryotes, the monomer size is shown to vary depending on the stage in the growth cycle. P. aurelia exhibits a monomer size of 153±7 bp and 178±6 bp and T. pyriformis 207±10 bp and 230±10 bp in logarithmic and stationary cells, respectively. Both exhibit a nucleosome size of 140 bp. We discuss the possible association of these changes with histone content and nuclear activity changes, and also a possible reason for the divergence from the size pattern of monomer repeats seen in lower eukaryotes by T. pyriformis .

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.