Epigenetic deregulation of lamina-associated domains in Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid disease characterized by the early onset of age-related phenotypes including arthritis, loss of body fat and hair, and atherosclerosis. Cells from affected individuals express a mutant version of the nuclear envelope protein lamin A (termed progerin) and have previously been shown to exhibit prominent histone modification changes. METHODS: Here, we analyze the possibility that epigenetic deregulation of lamina-associated domains (LADs) is involved in the molecular pathology of HGPS. To do so, we studied chromatin accessibility (Assay for Transposase-accessible Chromatin (ATAC)-see/-seq), DNA methylation profiles (Infinium MethylationEPIC BeadChips), and transcriptomes (RNA-seq) of nine primary HGPS fibroblast cell lines and six additional controls, two parental and four age-matched healthy fibroblast cell lines. RESULTS: Our ATAC-see/-seq data demonstrate that primary dermal fibroblasts from HGPS patients exhibit chromatin accessibility changes that are enriched in LADs. Infinium MethylationEPIC BeadChip profiling further reveals that DNA methylation alterations observed in HGPS fibroblasts are similarly enriched in LADs and different from those occurring during healthy aging and Werner syndrome (WS), another premature aging disease. Moreover, HGPS patients can be stratified into two different subgroups according to their DNA methylation profiles. Finally, we show that the epigenetic deregulation of LADs is associated with HGPS-specific gene expression changes. CONCLUSIONS: Taken together, our results strongly implicate epigenetic deregulation of LADs as an important and previously unrecognized feature of HGPS, which contributes to disease-specific gene expression. Therefore, they not only add a new layer to the study of epigenetic changes in the progeroid syndrome, but also advance our understanding of the disease's pathology at the cellular level.

Centromeric RNA and Its Function at and Beyond Centromeric Chromatin.

The most fundamental region of the chromosome for maintaining accurate genome segregation and stability is the centromeric chromatin [1,2]. In this review, we will focus on centromere-derived RNAs (cenRNAs), a crucial component of centromere structure first identified over 40 years ago [3] but only recently investigated in detail. Indeed, centromeric transcription is necessary for the proper formation of CENP-A-containing centromeric chromatin (cen-chromatin) [4-7] and for the formation of pericentromeric heterochromatin [8,9], and the transcripts play a role in the structure and function of the centromere-kinetochore interface [3,7,10]. Furthermore, cenRNA overexpression has been observed in some cancer patients [11-13] and has been shown to drive the formation of breast cancer in a mouse model system [14]. Together, these observations call for a more detailed appraisal of the composition, regulation and function of cenRNAs. In this review, we will first discuss the difficulties underlying the study of cenRNAs, then we identify different domains within the centromeric chromatin before we discuss the role of cenRNAs in the context of these domains.

Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex.

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

Investigating DNA supercoiling in eukaryotic genomes.

Supercoiling is a fundamental property of DNA, generated by polymerases and other DNA-binding proteins as a consequence of separating/bending the DNA double helix. DNA supercoiling plays a key role in gene expression and genome organization, but has proved difficult to study in eukaryotes because of the large, complex and chromatinized genomes. Key approaches to study DNA supercoiling in eukaryotes are (1) centrifugation-based or electrophoresis-based techniques in which supercoiled plasmids extracted from eukaryotic cells form a compacted writhed structure that migrates at a rate proportional to the level of DNA supercoiling; (2) in vivo approaches based on the preferential intercalation of psoralen molecules into under-wound DNA. Here, we outline the principles behind these techniques and discuss key discoveries, which have confirmed the presence and functional potential of unconstrained DNA supercoiling in eukaryotic genomes.

Effects of DNA supercoiling on chromatin architecture.

Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo.

Effects of DNA supercoiling on chromatin architecture.

Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo.

Profiling DNA supercoiling domains in vivo.

Transitions in DNA structure have the capacity to regulate genes, but have been poorly characterised in eukaryotes due to a lack of appropriate techniques. One important example is DNA supercoiling, which can directly regulate transcription initiation, elongation and coordinated expression of neighbouring genes. DNA supercoiling is the over- or under-winding of the DNA double helix, which occurs as a consequence of polymerase activity and is modulated by topoisomerase activity [5]. To map the distribution of DNA supercoiling in nuclei, we developed biotinylated 4,5,8-trimethylpsoralen (bTMP) pull-down to preferentially enrich for under-wound DNA. Here we describe in detail the experimental design, quality controls and analyses associated with the study by Naughton et al. [13] that characterised for the first time the large-scale distribution of DNA supercoiling in human cells (GEO: GSE43488 and GSE43450GSE43488GSE43450).

Divergent RNA transcription: a role in promoter unwinding?

New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance.

Polymers for the cell-specific immobilisation of megakaryocytic cell lines.

Primary human megakaryocytes, the precursor cells of platelets, are difficult to collect and cultivate. Polymers that enrich these cells without affecting their regulation or function are of interest for basic research as well as for cord blood transplantation purposes since co-transplantation of enriched megakaryocyte concentrates increase the success of stem cell therapy. Herein, polymer microarrays were used for the discovery of substrates for MEG-01 cells, with five polymers identified which selectively bound cells of the megakaryocytic lineage. Flow cytometry and miRNA profiling revealed that immobilisation had only a minor effect on the cellular maturation status, making the identified substrates potential candidates for concentrating megakaryocytes from patients prior to transplantation.

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures.

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling, we used biotinylated trimethylpsoralen as a DNA structure probe to show that the human genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF insulator protein-binding sites. Underwound domains are transcriptionally active and enriched in topoisomerase I, 'open' chromatin fibers and DNase I sites, but they are depleted of topoisomerase II. Furthermore, DNA supercoiling affects additional levels of chromatin compaction as underwound domains are cytologically decondensed, topologically constrained and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation, providing an evolutionary purpose for clustering genes along chromosomes.