Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.     

CTCF elements direct allele-specific undermethylation at the imprinted H19 locus

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.     

Role of CTCF binding sites in the Igf2/H19 imprinting control region

A approximately 2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis-the H19 promoter and gene-also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR.     

Nuclear envelope-limited chromatin sheets (ELCS) and heterochromatin higher order structure

The interphase nucleus and nuclear envelope can acquire a myriad of shapes in normal or pathological cell states. There exist a wide variety of indentations and invaginations, of protrusions and evaginations. It has been difficult to classify and name all of these nuclear shapes and, consequently, a barrier to understanding the biochemical and biophysical causes. This review focuses upon one type of nuclear envelope shape change, named “nuclear envelope-limited chromatin sheets” (ELCS), which appears to involve exaggerated nuclear envelope growth, carrying with it one or more layers of ∼30 nm diameter heterochromatin. A hypothesis on the formation of ELCS is proposed, relating higher order heterochromatin structure in an interphase nucleus, nuclear envelope growth, and nuclear envelope-heterochromatin interactions.     

A possible role of Drosophila CTCF in mitotic bookmarking and maintaining chromatin domains during the cell cycle

Background

The CCCTC-binding factor (CTCF) is a highly conserved insulator protein that plays various roles in many cellular processes. CTCF is one of the main architecture proteins in higher eukaryotes, and in combination with other architecture proteins and regulators, also shapes the three-dimensional organization of a genome. Experiments show CTCF partially remains associated with chromatin during mitosis. However, the role of CTCF in the maintenance and propagation of genome architectures throughout the cell cycle remains elusive.

Results

We performed a comprehensive bioinformatics analysis on public datasets of Drosophila CTCF (dCTCF). We characterized dCTCF-binding sites according to their occupancy status during the cell cycle, and identified three classes: interphase-mitosis-common (IM), interphase-only (IO) and mitosis-only (MO) sites. Integrated function analysis showed dCTCF-binding sites of different classes might be involved in different biological processes, and IM sites were more conserved and more intensely bound. dCTCF-binding sites of the same class preferentially localized closer to each other, and were highly enriched at chromatin syntenic and topologically associating domains boundaries.

Conclusions

Our results revealed different functions of dCTCF during the cell cycle and suggested that dCTCF might contribute to the establishment of the three-dimensional architecture of the Drosophila genome by maintaining local chromatin compartments throughout the whole cell cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0019-6) contains supplementary material, which is available to authorized users.     

Changes in chromatin structure during the mitotic cycle

Summary Optical density profiles of Feulgen-stained nuclei ofBryonia dioica at different stages of the mitotic cycle were determined. Nuclei in the G₂ phase have a greater fraction of dense chromatin than nuclei in G₁ phase. However, nuclei at the end of the S phase have dispersed chromatin of minimal density. Thus, chromatin density oscillates during the mitotic cycle of this species, consequently the progressive increase in density previously recorded throughout the intermitotic period of two other species (onion and mouse) cannot be a general rule.     

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.     

Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.     

Theory of H1-mediated control of higher orders of structure in chromatin

It is known that the lysine-rich histone H1 induces both higher orders of folding in chromatin and donut shapes in DNA. However, these phenomena occur only on the high-salt side of a narrow transition range located at about 0.02M salt. Previous theoretical analyses of the ionic-strength dependencies of DNA persistence length and denaturation rate have provided the information that the basic rigid-rod unit in high-molecular-weight DNA is a segment 60 base pairs in length and that if the phosphate charge is neutralized, this segment will spontaneously adopt a bent conformation with radius of curvature 170 Å. On the assumption that an H1 molecule does not completely neutralize the DNA charge in its vicinity, the theory has been extended here to determine the onset of spontaneous bending as a function of salt concentration and extent of phosphate neutralization. A salt transition of the kind observed has been found for the realistic value of 82% charge neutralization, with the actual value likely to be in the neighborhood of 90%, as suggested by the measurements of Wilson and Bloomfield.¹ It is recalled that the spacer DNA length in chromatin is of about the same length as the DNA rigid-rod unit. If binding of H1 to the spacer induces, as predicted, a bent conformation of radius about 170 Å, then the observed value of about 150 Å for the outer radius of the solenoid presently thought to be the basic mode of folding for a nucleosome chain can be understood as a reflection of the inherent maximum curvature of DNA in aqueous salt solution.     

A higher order chromatin structure that is lost during differentiation of mouse neuroblastoma cells

Application of differential scanning calorimetry to nuclei from rapidly growing mouse neuroblastoma cells showed a melting profile with four major thermal transitions: I (60 degrees C), II (76 degrees C), III (88 degrees C), and IV (105 degrees C). When neuroblastoma cells were induced to differentiate by serum withdrawal or treatment with sodium butyrate, transition IV disappeared, while transition III increased in magnitude. Comparison was made to nuclei from several types of nondividing cells as well as a number of samples from mature tissues. In rapidly dividing cells the predominant endotherm was IV (105 degrees C), while in nondividing cells, transition III (88 degrees C) predominated the calorimetric profile. Cellular differentiation thus appeared to be accompanied by a major change in chromatin structure, as evidenced by a shift in melting temperature from 105 to 90 degrees C, and this may serve to distinguish the Go phase of the cell cycle from G1.     

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.     

Content of histone H1 and histone phosphorylation in relation to the higher order structures of chromatin in Drosophila

The amount of histone H1 relative to core histones has been determined in three Drosophila species (D. melanogaster, D. texana and D. virilis) in chromatin from several tissues differing in chromatin structure and genetic activity. Low levels of H1 were found in relatively undifferentiated, early embryos as well as in a line of cultured cells. In late embryos the content of H1 was highest in D. virilis which possesses larger amounts of and a partially more compacted constitutive heterochromatin than the two other species. Polytene chromatin from larval salivary glands showed increased levels of H1 compared with diploid chromatin and the degree of phosphorylation of this histone was relatively low. The degree of phosphorylation of H2A was found to be drastically reduced in polytene as compared with diploid embryonic chromatin, which parallels the extensive underreplication of constitutive heterochromatin. Also, in diploid chromatin a qualitative correlation was observed between the relative amounts of heterochromatin and the levels of H2A phosphorylation. These findings suggest a connection between H2A phosphorylation and heavy compaction of interphase chromatin.     

Nuclear envelope-limited chromatin sheets are part of mitotic death

Nuclear envelope-limited chromatin sheets (ELCS) are enigmatic membranous structures of uncertain function. This study describes the induction of ELCS in p53 mutated Burkitt's lymphoma cell lines after treatment with irradiation or the microtubule inhibitor, SK&F 96365. Both treatments evoked similar mitotic death, involving metaphase arrest followed by extensive endopolyploidisation and delayed apoptosis, although the kinetics were different. We found that induction of ELCS and nuclear segmentation correlated with the amount and kinetics of M-phase arrest, mitosis restitution and delayed apoptosis of endopolyploid cells. In metaphases undergoing restitution, ELCS are seen participating in the restoration of the nuclear envelope, mediating the attachment of peripheral chromatin to it. In interphase cells, ELCS join nuclear segments, ectopically linking and fusing with heterochromatin regions. In cells with segmented nuclei, continued DNA replication was observed, along with activation and redistribution of Ku70, suggestive of non-homologous DNA end-joining. Induction of ELCS also parallels the induction of cytoplasmic stacked membrane structures, such as confronting cisternae and annulate lamellae, which participate in the turnover and degeneration of ELCS. The results suggest that arrest at a spindle checkpoint and the uncoupling of mitosis from DNA replication lead to the emergence of ELCS in the resulting endopolyploid cells.     

The DNase I sensitive state of active globin gene chromatin resists trypsin treatments which disrupt chromatin higher order structure

Active genes in higher eukaryotes reside in chromosomal domains which are more sensitive to digestion by DNase I than the surrounding inactive chromatin. Although it is widely assumed that some modification of higher order structure is important to the preferential DNase I sensitivity of active chromatin, this has so far not been tested. Here we show that the structural distinction between DNase I sensitive and resistant chromatin is remarkably stable to digestion by trypsin. Chick embryonic red blood cell nuclei were subjected to increasing levels of trypsin digestion and then assayed in the following three ways: (1) by gel electrophoresis for histone cleavage, (2) by sedimentation and nuclease digestion for loss of higher order structure, and (3) by dot-blot hybridization to globin and ovalbumin probes for disappearance of preferential DNase I sensitivity. We have found that chromatin higher order structure is lost concomitantly with the cleavage of histones H1, H5, and H3. In contrast, the preferential sensitivity of the globin domain to DNase I persists until much higher concentrations of trypsin, and indeed is not completely abolished even by the highest levels of trypsin we have used. We therefore conclude that the structural distinction of active chromatin, recognized by DNase I, does not reside at the level of higher order structure.     

Periodicity of DNA folding in higher order chromatin structures.

Each level of DNA folding in cells corresponds to a distinct chromatin structure. The basic chromatin units, nucleosomes, are arranged into solenoids which form chromatin loops. To characterize better the loop organization of chromatin we have assumed that the accessibility of DNA inside these structures is lower than on the outside and examined the size distribution of high mol. wt DNA fragments obtained from cells and isolated nuclei after digestion with endogenous nuclease or topoisomerase II. The largest discrete fragments obtained contain 300 kbp of DNA. Their further degradation proceeds through another discrete size step of 50 kbp. This suggests that chromatin loops contain approximately 50 kbp of DNA and that they are grouped into hexameric rosettes at the next higher level of chromatin structure. Based upon these observations a model by which the 30 nm chromatin fibre can be folded up into compact metaphase chromosomes is also described.