The human X-linked macrosatellite DXZ4 is a large tandem repeat located at Xq23 that is packaged into heterochromatin on the male X chromosome and female active X chromosome and, in response to X chromosome, inactivation is organized into euchromatin bound by the insulator protein CCCTC-binding factor (CTCF) on the inactive X chromosome (Xi). The purpose served by this unusual epigenetic regulation is unclear, but suggests a Xi-specific gain of function for DXZ4. Other less extensive bands of euchromatin can be observed on the Xi, but the identity of the underlying DNA sequences is unknown. Here, we report the identification of two novel human X-linked tandem repeats, located 58 Mb proximal and 16 Mb distal to the macrosatellite DXZ4. Both tandem repeats are entirely contained within the transcriptional unit of novel spliced transcripts. Like DXZ4, the tandem repeats are packaged into Xi-specific CTCF-bound euchromatin. These sequences undergo frequent CTCF-dependent interactions with DXZ4 on the Xi, implicating DXZ4 as an epigenetically regulated Xi-specific structural element and providing the first putative functional attribute of a macrosatellite in the human genome.

BackgroundThe X-linked macrosatellite DXZ4 is a large homogenous tandem repeat that in females adopts an alternative chromatin organization on the primate X chromosome in response to X-chromosome inactivation. It is packaged into heterochromatin on the active X chromosome but into euchromatin and bound by the epigenetic organizer protein CTCF on the inactive X chromosome. Because its DNA sequence diverges rapidly beyond the New World monkeys, the existence of DXZ4 outside the primate lineage is unknown.ResultsHere we extend our comparative genome analysis and report the identification and characterization of the mouse homolog of the macrosatellite. Furthermore, we provide evidence of DXZ4 in a conserved location downstream of the PLS3 gene in a diverse group of mammals, and reveal that DNA sequence conservation is restricted to the CTCF binding motif, supporting a central role for this protein at this locus. However, many features that characterize primate DXZ4 differ in mouse, including the overall size of the array, the mode of transcription, the chromatin organization and conservation between adjacent repeat units of DNA sequence and length. Ctcf binds Dxz4 but is not exclusive to the inactive X chromosome, as evidenced by association in some males and equal binding to both X chromosomes in trophoblast stem cells.ConclusionsCharacterization of Dxz4 reveals substantial differences in the organization of DNA sequence, chromatin packaging, and the mode of transcription, so the potential roles performed by this sequence in mouse have probably diverged from those on the primate X chromosome.

The nucleolus is a nuclear compartment that plays an important role in ribosome biogenesis. Some structural features and epigenetic patterns are shared between nucleolar and non-nucleolar compartments. For example, the location of transcriptionally active mRNA on extended chromatin loop species is similar to that observed for transcriptionally active ribosomal DNA (rDNA) genes on so-called Christmas tree branches. Similarly, nucleolus organizer region-bearing chromosomes located a distance from the nucleolus extend chromatin fibers into the nucleolar compartment. Specific epigenetic events, such as histone acetylation and methylation and DNA methylation, also regulate transcription of both rRNA- and mRNA-encoding loci. Here, we review the epigenetic mechanisms and structural features that regulate transcription of ribosomal and mRNA genes. We focus on similarities in epigenetic and structural regulation of chromatin in nucleoli and the surrounding non-nucleolar region and discuss the role of proteins, such as heterochromatin protein 1, fibrillarin, nucleolin, and upstream binding factor, in rRNA synthesis and processing.

Heterochromatin protein 1 (HP1), which binds to sites of histone H3 lysine 9 (H3K9) methylation, is primarily responsible for gene silencing and the formation of heterochromatin. We observed that HP1 beta is located in both the chromocenters and fibrillarin-positive nucleoli interiors. However, HP1 alpha and HP1 gamma occupied fibrillarin-positive compartments to a lesser extent, corresponding to the distinct levels of HP1 subtypes at the promoter of rDNA genes. Deficiency of histone methyltransferases SUV39h and/or inhibition of histone deacetylases (HDACi) decreased HP1 beta and H3K9 trimethylation at chromocenters, but not in fibrillarin-positive regions that co-localized with RNA polymerase I. Similarly, SUV39h- and HDACi-dependent nucleolar rearrangement and inhibition of rDNA transcription did not affect the association between HP1 beta and fibrillarin. Moreover, the presence of HP1 beta in nucleoli is likely connected with transcription of ribosomal genes and with the role of fibrillarin in nucleolar processes.