Pericentromeric heterochromatin domains are maintained without accumulation of HP1.

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1alpha or HP1beta proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1alpha, HP1beta, and HP1gamma in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1alpha, HP1beta, and HP1gamma from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1alpha, HP1beta, and HP1gamma at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains.

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Large-scale chromatin organization and the localization of proteins involved in gene expression in human cells.

Compartmentalization of the interphase nucleus is an important element in the regulation of gene expression. Here we investigated the functional organization of the interphase nucleus of HeLa cells and primary human fibroblasts. The spatial distribution of proteins involved in transcription (TFIIH and RNA polymerase II) and RNA processing and packaging (hnRNP-U) were analyzed in relation to chromosome territories and large-scale chromatin organization. We present evidence that these proteins are present predominantly in the interchromatin space, inside and between chromosome territories, and are largely excluded by domains of condensed chromatin. We show that they are present throughout the active and inactive X-chromosome territories in primary female fibroblasts, indicating that these proteins can freely diffuse throughout the interchromatin compartment in the interphase nucleus. Furthermore, we established that the in vivo spatial distribution of condensed chromatin in the interphase nucleus does not depend on ongoing transcription. Our data support a conceptually simple model for the functional organization of interphase nuclei.

Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location.

Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. We performed large-scale mapping and analysis of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, we identified approximately 100-200 target genes among >6000 probed loci. We found that HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from those of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes.

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules.

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place primarily at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors.