Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG–binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG–binding domain (MBD) only. Similar to MeCP2, another methyl CpG–binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.

A quantitative comparison of higher-order chromatin arrangements was performed in human cell types with three-dimensionally (3D) preserved, differently shaped nuclei. These cell types included flat-ellipsoid nuclei of diploid amniotic fluid cells and fibroblasts and spherical nuclei of B and T lymphocytes from peripheral human blood. Fluorescence in-situ hybridization (FISH) was performed with chromosome paint probes for large (#1-5) and small (#17-20) autosomes, and for the two sex chromosomes. Other probes delineated heterochromatin blocks of numerous larger and smaller human chromosomes. Shape differences correlated with distinct differences in higher order chromatin arrangements: in the spherically shaped lymphocyte nuclei we noted the preferential positioning of the small, gene dense #17, 19 and 20 chromosome territories (CTs) in the 3D nuclear interior--typically without any apparent connection to the nuclear envelope. In contrast, CTs of the gene-poor small chromosomes #18 and Y were apparently attached at the nuclear envelope. CTs of large chromosomes were also preferentially located towards the nuclear periphery. In the ellipsoid nuclei of amniotic fluid cells and fibroblasts, all tested CTs showed attachments to the upper and/or lower part of the nuclear envelope: CTs of small chromosomes, including #18 and Y, were located towards the centre of the nuclear projection (CNP), while the large chromosomes were positioned towards the 2D nuclear rim. In contrast to these highly reproducible radial arrangements, 2D distances measured between heterochromatin blocks of homologous and heterologous CTs were strikingly variable. These results as well as CT painting let us conclude that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs. 3D-modelling of statistical arrangements of 46 human CTs in spherical nuclei was performed under the assumption of a linear correlation between DNA content of each chromosome and its CT volume. In a set of modelled nuclei, we noted the preferential localization of smaller CTs towards the 3D periphery and of larger CTs towards the 3D centre. This distribution is in clear contrast to the experimentally observed distribution in lymphocyte nuclei. We conclude that presently unknown factors (other than topological constraints) may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.

Background: Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals.

There is increasing evidence of crosstalk between epigenetic modifications such as histone and DNA methylation, recognized by HP1 and methyl CpG-binding proteins, respectively. We have previously shown that the level of methyl CpG-binding proteins increased dramatically during myogenesis leading to large-scale heterochromatin reorganization. In this work, we show that the level of HP1 isoforms did not change significantly throughout myogenic differentiation but their localization did. In particular, HP1γ relocalization to heterochromatin correlated with MeCP2 presence. Using co-immunoprecipitation assays, we found that these heterochromatic factors interact in vivo via the chromo shadow domain of HP1 and the first 55 amino acids of MeCP2. We propose that this dynamic interaction of HP1 and MeCP2 increases their concentration at heterochromatin linking two major gene silencing pathways to stabilize transcriptional repression during differentiation.