4. Discussion
4.4. Epigenetic changes have a limited direct effect on gene expression
sites. At the same time, the relatively long expression window of 48 hours may have contributed to generating false-positive findings.
Third, the data obtained are specific to cultured dermal fibroblasts. As with the rest of the results obtained in this work, their scope is limited and should be confirmed in other cell types and primary tissues to better evaluate their role in the pathology of the disease.
Fourth, and most importantly, it is critical to remember that the LAD data generated herein represent population averages from a large number of cells. On the one hand, this ensures the comparability with other population-scale data produced in this work, but, on the other hand, it probably underestimates the extent of LAD alterations occurring in the highly heterogeneous HGPS fibroblast population. Assuming that the integration of Progerin obeys at least some degree of stochasticity, a significant number of alterations are likely being missed due to an averaging over the entire cell population. Future studies will allow the study of single-cell LAD interactomes, which, in combination with other single-cell data, will not only give a better overview of the extent of aberrations in HGPS cells but also allow a better understanding of how the nuclear envelope-chromatin interplay contributes to the pathology of the disease.
4.4. Epigenetic changes have a limited direct effect on gene
expression
Aside from DNA methylation, chromatin accessibility and changes in the LAD landscape, alterations at the transcriptomic level were analyzed in this work. The obtained results highlight a substantial deregulation of gene expression in HGPS fibroblasts, including an enrichment of pathways associated with key pathological features as well as an aggravation in cells from older patients.
Gene expression in progeria fibroblasts has been profiled before (Ly et al., 2000; Park et al., 2001; Csoka et al., 2004; Kubben et al., 2016). Importantly, the data generated in this work were in good correlation with those of previous genome-scale expression studies, especially for more strongly deregulated genes. In addition, they confirmed the deregulation of developmental, morphological, ECM- and cardiovascular function-related processes, as well as the central role of the transcriptional regulators NRF2 and AP1 in the disease (Csoka et al., 2004; Pereira et al., 2008; Marji et al., 2010; Kubben et al., 2016). A comparison with a more recent study establishing a transcriptome-based age predictor using HGPS fibroblasts (Fleischer et al., 2018) was not performed. However, the fact that an age increase of about 10 years was identified for
4. Discussion 4.4 Epigenetic changes have a limited direct effect on gene expression
HGPS cells in the same study (Fleischer et al., 2018), which closely matches the DNA methylation-based age acceleration identified herein (9.73 years), suggests a good overlap with that work, as well.
One key benefit of the expansive sample set available for the present study is that gene expression profiles from HGPS samples of different age groups could be analyzed. In general, the finding that fibroblasts from older patients show an enrichment of pathology-related features is expected, as symptoms progressively worsen with advanced patient age (Merideth et al., 2008). For instance, HGPS patients are affected by alopecia and severe skin abnormalities including changes in pigmentation, skin dimpling, loss of subcutaneous fat and the development of sclerotic skin (Merideth et al., 2008; Rork et al., 2014). The observed deregulation of core dermal fibroblast functions, such as ECM maintenance and collagen production (Sorrell and Caplan, 2004), is therefore unsurprising and may actually contribute to the skin phenotype in HGPS patients. More intriguing is the observation that HGPS fibroblasts show a deregulation of genes involved in cardiovascular development, i.e., a function not classically associated with dermal fibroblasts. Crucially, the differential expression of these genes was found to be correlated with differences in Lamin A-binding, thus offering a potential mechanistic explanation for the activation of lineage-unspecific genes, which may be conserved in other cell types.
In this respect, one of the central findings of this work is that an activation of non-dermal fibroblast-specific genes, through the redistribution of the LAD landscape in Progerin-expressing cells, contributes to the HGPS-related transcriptome changes. Conceptually, this conclusion confirms the central role that Lamin A mutations play in the reconfiguration of the LAD and chromatin layers in laminopathies (Briand and Collas, 2018). In fact, a similar mechanism, including an induction of lineage-unspecific genes, has recently been described for fibroblasts carrying a LMNA mutation that causes Emery-Dreifuss muscular dystrophy (Perovanovic et al., 2016). Additionally, mutations in the LMNA gene lead to a drastic redistribution of the LAD landscape and accompanying gene expression changes in cardiac myocytes from dilated cardiomyopathy patients (Cheedipudi et al., 2019). Given this evidence, it is tempting to speculate that the same mechanism may also contribute to the severe pathology affecting other Progerin-expressing cell types such as VSMCs and endothelial cells. In this respect, it has to be noted, however, that Progerin expression in endothelial cells has recently been reported to cause cardiovascular pathology more directly through an impaired mechanoresponse (Osmanagic-Myers et al., 2018). In this work, the authors showed that Progerin-induced changes in mechanosignaling at the nuclear envelope contribute to excessive fibrosis and the
4. Discussion 4.4 Epigenetic changes have a limited direct effect on gene expression
future experiments with primary, non-fibroblast cell populations and HGPS in vivo models will be necessary to better define the role of these mechanisms in the disease.
Interestingly, the gene expression changes were only partially reflected by alterations at the chromatin accessibility and DNA methylation level in HGPS fibroblasts. In other words, the limited number of differentially expressed genes with simultaneous HGPS-specific differences in DNA methylation or chromatin accessibility appears surprising. A number of technical and biological factors may have contributed to this.
From a technical perspective, simple biological variation may have limited the effect size, as a relatively small number of samples was used for the analyses, with some HGPS samples not overlapping between different experiments. Likewise, many of the fibroblast samples were obtained from different sites of the body. Given that fibroblasts are a highly heterogeneous cell type (Sorrell and Caplan, 2004; Driskell and Watt, 2015), lineage-specific differences between the fibroblast populations may thus have hindered the detection of further HGPS-related epigenetic alterations.
From a biological perspective, the extensive population heterogeneity of HGPS cells may have had a strong impact on the observed effect size. More precisely, the ATAC-see and Lamin A/C immunofluorescence experiments revealed high degrees of cell-to-cell variation regarding nuclear malformation and chromatin accessibility in the fibroblasts. This probably limited the number of regions identified as significantly differentially accessible, which represent a consensus shared by a certain number of Progerin-expressing cells in the population. Future single-cell-based studies utilizing single-cell ATAC-seq, single-cell RNA-seq and combinatorial methods will allow us to better capture the scope of epigenetic alterations in HGPS cells and their effect on individual transcriptomes.
Another potential explanation for the relatively small effect size comes from the fact that LADs are generally gene-poor (Guelen et al., 2008; van Steensel and Belmont, 2017). An accumulation of epigenetic changes in these regions may hence have little direct impact on the gene expression patterns in affected cells. But, as LADs function in the 3D organization of the genome, changes in the peripheral architecture can have consequences deep inside the nuclear interior. For example, Zheng and colleagues recently demonstrated that a knockout of all lamins in mouse embryonic stem cells leads to global transcriptional alterations not restricted to genes in LADs (Zheng et al., 2018). Mechanistically, such changes can be catalyzed through altered interactions between topologically-associated chromatin domains (Zheng et al., 2018) or