The end adjusts the means: Heterochromatin remodelling during terminal cell differentiation

All cells that constitute mature tissues in an eukaryotic organism undergo a multistep process of cell differentiation. At the terminal stage of this process, cells either cease to proliferate forever or rest for a very long period of time. During terminal differentiation, most of the genes that are required for cell ‘housekeeping’ functions, such as proto-oncogenes and other cell-cycle and cell proliferation genes, become stably repressed. At the same time, nuclear chromatin undergoes dramatic morphological and structural changes at the higher-order levels of chromatin organization. These changes involve both constitutively inactive chromosomal regions (constitutive heterochromatin) and the formerly active genes that become silenced and structurally modified to form facultative heterochromatin. Here we approach terminal cell differentiation as a unique system that allows us to combine biochemical, ultrastructural and molecular genetic techniques to study the relationship between the hierarchy of chromatin higher-order structures in the nucleus and its function(s) in dynamic packing of genetic material in a form that remains amenable to regulation of gene activity and other DNA-dependent cellular processes.     

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]₄, a microsatellite that also forms part of the pericentromeres, together with [GA]₈, [GATA]₄ and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.     

Cell-Growth Kinetics and Karyotype Analysis of Human Memory Lymphocytes Responding to Alloantigen and Mitogen

Peripheral-blood lymphocytes were primed in vitro with the mitogen phytohemagglutinin (PHA) or with allogeneic cells and their memory responses studied following sequential restimulation with either mitogen or alloantigen. Chromosome preparations were made every 12 hours following exposure to the stimulating agents. Cultures were labeled with BUdR for sister-chromatid staining of the chromosomes which provided information about the kinetics of cell growth and rates of sister chromatid exchange. Cultures containing no BUdR were used for the investigation of cell karyotypes after chromosome-banding.Following PHA as well as alloantigen restimulation, an earlier reaction of the responding cells was observed. The peak response after the first stimulation was found at 120 h with allogeneic stimulation and at 60 h with mitogen stimulation. In the second round of stimulation, the peak occurred after 48 h (allogeneic) and 36 h (PHA) and following the third stimulation after 36 h (allogeneic) and 24 h (PHA). The speed of cell growth was decreased following restimulation with either alloantigen and mitogen. In contrast to the allogeneic restimulation, the number of cells responding after PHA restimulation was decreased.No systematic numerical or structural aberration of the karyotype was detected following repeated stimulation with either alloantigen or mitogen. In this sense, the lymphocyte subpopulations selected by repeated stimulation did not differ from the starting material. On the other hand, the sister-chromatid exchange (SCE) frequency was increased following allogeneic restimulation, whereas it remained constant with PHA restimulation.     

Complex evolution of X and Y autosomal translocations in the giant mole-rat, Cryptomys mechowi (Bathyergidae)

Cross-species chromosome painting was used to determine homologous chromosomal regions between two species of mole-rat, the naked mole-rat, Heterocephalus glaber (2n = 60), and the giant mole-rat, Cryptomys mechowi (2n = 40), using flow-sorted painting probes representative of all but two of the H. glaber chromosomal complement. In total 43 homologous regions were identified in the C. mechowi genome. Eight H. glaber chromosomes are retained in toto in C. mechowi, and 13 produce two or more signals in this species. The most striking difference in the karyotypes of the two taxa concerns their sex chromosomes. The H. glaber painting probes identified a complex series of translocations that involved the fractionation of four autosomes and the subsequent translocation of segments to the sex chromosomes and to autosomal partners in the C. mechowi genome. An intercalary heterochromatic block (IHB) was detected in sex chromosomes of C. mechowi at the boundary with the translocated autosomal segment. We discuss the likely sequence of evolutionary events that has led to the contemporary composition of the C. mechowi sex chromosomes, and consider these in the light of prevailing views on the genesis of sex chromosomes in mammals.     

Methylation of a euchromatin-heterochromatin transition region in Arabidopsis thaliana chromosome 5 left arm

Cytosine methylation was studied at the level of the euchromatin/heterochromatin transition genomic region of the Arabidopsis chromosome 5 left arm. It has been shown using a monoclonal antibody against 5-methylcytosines that the density of DNA methylation increases from the euchromatin towards the heterochromatin. YACs mapped along this region were characterized for their repeated sequences content. Some of them, corresponding to euchromatin, euchromatin/heterochromatin border and heterochromatin regions, were used as probes for a Southern blot analysis of methylation. This revealed that the degree of mCmCGG and GATmC methylation increases significantly from the euchromatin towards the heterochromatin. Moreover, an analysis of cytosine methylation levels (% of 5-methylcytosine) of different DNA fragments, inside the same genomic region, was performed using PCR and/or Southern blot approaches. There is a gradual increase of methylation along the genomic region analyzed: CpG methylation in the euchromatic fraction, CpG and CpNpG methylation at the euchromatin/heterochromatin transition and an additional asymmetrical methylation in the repeated-heterochromatic fraction. The most methylated repeated family at CpG, CpNpG and asymmetrical sites is the 5S ribosomal DNA, highly methylated even though it is transcribed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68–127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear organization of centromeric domains is not perturbed by inhibition of histone deacetylases

It is well established that modification of lysines in histone molecules correlates with gene expression and chromatin structure. It is not known whether this operates entirely at a local level, e.g. through the recruitment of specific proteins, or whether histone modifications might impact on more long-range aspects of chromatin organization. There is a distinctive organization of chromatin within the nucleus and the chromatin at the nuclear periphery of mammalian cells appears to be hypoacetylated. Previously it had been suggested that inhibition of histone deacetylases by TSA causes a gross remodeling of nuclear structure, specifically the recruitment of centromeric heterochromatin to the nuclear periphery. Here, we have quantified the nuclear organization of histone modifications and the localization of centromeric domains in human cells before and after TSA treatment. TSA alters the nuclear distribution of histone acetylation, but not that of histone methylation. TSA elevates levels of histone acetylation at the nuclear periphery but we see no alteration in the position of centromeric domains in the nuclei of treated cells. We conclude that the distinctive nuclear localization of centromeric domains is independent of histone acetylation.