The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1

The organization of chromatin in the nucleus is nonrandom. Different genomic regions tend to reside in preferred nuclear locations, relative to radial position and nuclear compartments. Several lines of evidence support a role for chromatin localization in the regulation of gene expression. Therefore, a key problem is how the organization of chromatin is established and maintained in dividing cell populations. There is controversy about the extent to which chromatin organization is inherited from mother to daughter nucleus. We have used time-lapse microscopy to track specific human loci after exit from mitosis. In comparison to later stages of interphase, we detect increased chromatin mobility during the first 2 hr of G1, and during this period association of loci with nuclear compartments is both gained and lost. Although chromatin in daughter nuclei has a rough symmetry in its spatial distribution, we show, for the first time, that the association of loci with nuclear compartments displays significant asymmetry between daughter nuclei and therefore cannot be inherited from the mother nucleus. We conclude that the organization of chromatin in the nucleus is not passed down precisely from one cell to its descendents but is more plastic and becomes refined during early G1.     

Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line

Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.     

The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation.     

DNA distribution in fractions differing in the strength of association with nuclear matrix during activation of DNA endonucleases in cell nuclei and treatment with nuclease S1

Influence of activation of Ca2+/Mg2(+)-dependent, Mn2(+)-dependent, Mg2(+)-dependent and acidic endogenous DNAses on distribution of DNA in fractions differing in tightness of association with the nuclear matrix has been investigated. In the intact cell nuclei all types of DNA-protein bonds were obscured by a tight bonding of DNA with the proteins of replicative complex. Activation of endogenous nuclease activities caused detachment of a significant chromatin fraction from the nuclear matrix, fraction of DNA remained attached to the replicative complex, small fraction of DNA was bound to the nuclear matrix with a less tight bond. The endogenous nucleases are supposed to make cuts mainly in distal parts of the chromatin loops and do not affect the replicative complex, where the tight DNA-matrix bond is localized. Single-strand DNA-specific S1-nuclease preferably attacks the latter site.     

Localization of HPV-16 DNA sequence in CaSki cells by electron microscopic hybridocytochemistry.

We investigated the HPV-16 DNA sequence in the CaSki cervical carcinoma cell line by electron microscopic hybridocytochemistry using biotinylated HPV-16/18 probes. At the light microscopic level, reaction product of hybridized HPV-16 DNA sequence was not seen in the cytoplasm but appeared as spots or rods randomly distributed in the nuclei. By electron microscopy, reaction product was seen aggregated in several regions in the nuclei. Most of the stained areas did not reveal particular architecture but showed part of the chromatin structure. In other nuclei, reaction product was observed to be associated with strings of loop-like structure, and some stained loops were seen to be connected directly to the nuclear filamentous chromatin structure. The skeletonized images of hybridized HPV-16 DNA in the nuclei were illustrated by computerized image analysis. In conclusion, we have demonstrated the HPV-16 DNA sequence in the nuclei of CaSki cells by electron microscopy. The identification of stained areas localized only in the chromatin suggests an integrated form of HPV-16 DNA sequence in the cells. This method could be used to identify an integrated or episomal form of viral DNA in the virus-containing cells.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.     

Characterization of an Mg2+-dependent endonucleolytic activity of the rat hepatocyte nuclear matrix

Initial degradation of chromatin into high-molecular mass DNA fragments during apoptosis reflects the periodicity of chromatin organization into nuclear matrix-attached loops. In this article, we put forward the hypothesis that this pattern of DNA cleavage is also a result of the localization of an endonuclease on the nuclear matrix. Namely, we observed an endonucleolytic activity of the isolated rat hepatocyte nuclear matrix. It was Mg2+-dependent, with an optimal activity at pH 7.2 in the absence of either Na+ or K+. It was fully active in the presence of Zn2+ and capable of introducing single-strand breaks into plasmid DNA. It did not display a sequence-specific activity. A 23 kDa DNA nuclease that was principally localized on the rat hepatocyte nuclear matrix was detected. The enzyme shared the biochemical requirements with the nuclear matrix endonucleolytic activity, thus we proposed that p23 could be responsible for the endonucleolytic activity of the nuclear matrix. In view of its properties and preferential localization on the nuclear matrix, the endonuclease described herein could be a possible candidate that brings about initial DNA cleavage during apoptosis.     

Three-dimensional positioning of genes in mouse cell nuclei

To understand the regulation of the genome, it is necessary to understand its three-dimensional organization in the nucleus. We investigated the positioning of eight gene loci on four different chromosomes, including the β-globin gene, in mouse embryonic stem cells and in in vitro differentiated macrophages by fluorescence in situ hybridization on structurally preserved nuclei, confocal microscopy, and 3D quantitative image analysis. We found that gene loci on the same chromosome can significantly differ from each other and from their chromosome territory in their average radial nuclear position. Radial distribution of a given gene locus can change significantly between cell types, excluding the possibility that positioning is determined solely by the DNA sequence. For the set of investigated gene loci, we found no relationship between radial distribution and local gene density, as it was described for human cell nuclei. We did find, however, correlation with other genomic properties such as GC content and certain repetitive elements such as long terminal repeats or long interspersed nuclear elements. Our results suggest that gene density itself is not a driving force in nuclear positioning. Instead, we propose that other genomic properties play a role in determining nuclear chromatin distribution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Generating single-copy nuclear gene data for a recent adaptive radiation

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.     

Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach

Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.     

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.