Cancer, metastasis, and the epigenome.

Cancer is the second leading cause of death worldwide and disease burden is expected to increase globally throughout the next several decades, with the majority of cancer-related deaths occurring in metastatic disease. Cancers exhibit known hallmarks that endow them with increased survival and proliferative capacities, frequently as a result of de-stabilizing mutations. However, the genomic features that resolve metastatic clones from primary tumors are not yet well-characterized, as no mutational landscape has been identified as predictive of metastasis. Further, many cancers exhibit no known mutation signature. This suggests a larger role for non-mutational genome re-organization in promoting cancer evolution and dissemination. In this review, we highlight current critical needs for understanding cell state transitions and clonal selection advantages for metastatic cancer cells. We examine links between epigenetic states, genome structure, and misregulation of tumor suppressors and oncogenes, and discuss how recent technologies for understanding domain-scale regulation have been leveraged for a more complete picture of oncogenic and metastatic potential.

Topologically associating domains are stable units of replication-timing regulation.

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

A comparative encyclopedia of DNA elements in the mouse genome.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation.

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

RNA sequencing identifies common pathways between cigarette smoke exposure and replicative senescence in human airway epithelia.

BACKGROUND: Aging is affected by genetic and environmental factors, and cigarette smoking is strongly associated with accumulation of senescent cells. In this study, we wanted to identify genes that may potentially be beneficial for cell survival in response to cigarette smoke and thereby may contribute to development of cellular senescence. RESULTS: Primary human bronchial epithelial cells from five healthy donors were cultured, treated with or without 1.5% cigarette smoke extract (CSE) for 24 h or were passaged into replicative senescence. Transcriptome changes were monitored using RNA-seq in CSE and non-CSE exposed cells and those passaged into replicative senescence. We found that, among 1534 genes differentially regulated during senescence and 599 after CSE exposure, 243 were altered in both conditions, representing strong enrichment. Pathways and gene sets overrepresented in both conditions belonged to cellular processes that regulate reactive oxygen species, proteasome degradation, and NF-κB signaling. CONCLUSIONS: Our results offer insights into gene expression responses during cellular aging and cigarette smoke exposure, and identify potential molecular pathways that are altered by cigarette smoke and may also promote airway epithelial cell senescence.

Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia.

Abnormal replication timing has been observed in cancer but no study has comprehensively evaluated this misregulation. We generated genome-wide replication-timing profiles for pediatric leukemias from 17 patients and three cell lines, as well as normal B and T cells. Nonleukemic EBV-transformed lymphoblastoid cell lines displayed highly stable replication-timing profiles that were more similar to normal T cells than to leukemias. Leukemias were more similar to each other than to B and T cells but were considerably more heterogeneous than nonleukemic controls. Some differences were patient specific, while others were found in all leukemic samples, potentially representing early epigenetic events. Differences encompassed large segments of chromosomes and included genes implicated in other types of cancer. Remarkably, differences that distinguished leukemias aligned in register to the boundaries of developmentally regulated replication-timing domains that distinguish normal cell types. Most changes did not coincide with copy-number variation or translocations. However, many of the changes that were associated with translocations in some leukemias were also shared between all leukemic samples independent of the genetic lesion, suggesting that they precede and possibly predispose chromosomes to the translocation. Altogether, our results identify sites of abnormal developmental control of DNA replication in cancer that reveal the significance of replication-timing boundaries to chromosome structure and function and support the replication domain model of replication-timing regulation. They also open new avenues of investigation into the chromosomal basis of cancer and provide a potential novel source of epigenetic cancer biomarkers.

Chromatin-interaction compartment switch at developmentally regulated chromosomal domains reveals an unusual principle of chromatin folding.

Several 400- to 800-kb murine chromosome domains switch from early to late replication during loss of pluripotency, accompanied by a stable form of gene silencing that is resistant to reprogramming. We found that, whereas enhanced nuclease accessibility correlated with early replication genome-wide, domains that switch replication timing during differentiation were exceptionally inaccessible even when early-replicating. Nonetheless, two domains studied in detail exhibited substantial changes in transcriptional activity and higher-order chromatin unfolding confined to the region of replication timing change. Chromosome conformation capture (4C) data revealed that in the unfolded state in embryonic stem cells, these domains interacted preferentially with the early-replicating chromatin compartment, rarely interacting even with flanking late-replicating domains, whereas after differentiation, these same domains preferentially associated with late-replicating chromatin, including flanking domains. In both configurations they retained local boundaries of self-interaction, supporting the replication domain model of replication-timing regulation. Our results reveal a principle of developmentally regulated, large-scale chromosome folding involving a subnuclear compartment switch of inaccessible chromatin. This unusual level of regulation may underlie resistance to reprogramming in replication-timing switch regions.

Replication-timing boundaries facilitate cell-type and species-specific regulation of a rearranged human chromosome in mouse.

In multicellular organisms, developmental changes to replication timing occur in 400-800 kb domains across half the genome. While examples of epigenetic control of replication timing have been described, a role for DNA sequence in mammalian replication-timing regulation has not been substantiated. To assess the role of DNA sequences in directing developmental changes to replication timing, we profiled replication timing in mice carrying a genetically rearranged Human Chromosome 21 (Hsa21). In two distinct mouse cell types, Hsa21 sequences maintained human-specific replication timing, except at points of Hsa21 rearrangement. Changes in replication timing at rearrangements extended up to 900 kb and consistently reconciled with the wild-type replication pattern at developmental boundaries of replication-timing domains. Our results are consistent with DNA sequence-driven regulation of Hsa21 replication timing during development and provide evidence that mammalian chromosomes consist of multiple independent units of replication-timing regulation.

Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types.

To identify evolutionarily conserved features of replication timing and their relationship to epigenetic properties, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two human induced pluripotent stem cell lines (hiPSCs), and compared them with related mouse cell types. Results confirm the conservation of coordinately replicated megabase-sized "replication domains" punctuated by origin-suppressed regions. Differentiation-induced replication timing changes in both species occur in 400- to 800-kb units and are similarly coordinated with transcription changes. A surprising degree of cell-type-specific conservation in replication timing was observed across regions of conserved synteny, despite considerable species variation in the alignment of replication timing to isochore GC/LINE-1 content. Notably, hESC replication timing profiles were significantly more aligned to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Comparison with epigenetic marks revealed a signature of chromatin modifications at the boundaries of early replicating domains and a remarkably strong link between replication timing and spatial proximity of chromatin as measured by Hi-C analysis. Thus, early and late initiation of replication occurs in spatially separate nuclear compartments, but rarely within the intervening chromatin. Moreover, cell-type-specific conservation of the replication program implies conserved developmental changes in spatial organization of chromatin. Together, our results reveal evolutionarily conserved aspects of developmentally regulated replication programs in mammals, demonstrate the power of replication profiling to distinguish closely related cell types, and strongly support the hypothesis that replication timing domains are spatially compartmentalized structural and functional units of three-dimensional chromosomal architecture.

Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.

Differentiation of mouse embryonic stem cells (mESCs) is accompanied by changes in replication timing. To explore the relationship between replication timing and cell fate transitions, we constructed genome-wide replication-timing profiles of 22 independent mouse cell lines representing 10 stages of early mouse development, and transcription profiles for seven of these stages. Replication profiles were cell-type specific, with 45% of the genome exhibiting significant changes at some point during development that were generally coordinated with changes in transcription. Comparison of early and late epiblast cell culture models revealed a set of early-to-late replication switches completed at a stage equivalent to the post-implantation epiblast, prior to germ layer specification and down-regulation of key pluripotency transcription factors [POU5F1 (also known as OCT4)/NANOG/SOX2] and coinciding with the emergence of compact chromatin near the nuclear periphery. These changes were maintained in all subsequent lineages (lineage-independent) and involved a group of irreversibly down-regulated genes, at least some of which were repositioned closer to the nuclear periphery. Importantly, many genomic regions of partially reprogrammed induced pluripotent stem cells (piPSCs) failed to re-establish ESC-specific replication-timing and transcription programs. These regions were enriched for lineage-independent early-to-late changes, which in female cells included the inactive X chromosome. Together, these results constitute a comprehensive "fate map" of replication-timing changes during early mouse development. Moreover, they support a model in which a distinct set of replication domains undergoes a form of "autosomal Lyonization" in the epiblast that is difficult to reprogram and coincides with an epigenetic commitment to differentiation prior to germ layer specification.

Replication timing: a fingerprint for cell identity and pluripotency.

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.

G9a selectively represses a class of late-replicating genes at the nuclear periphery.

We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

Global reorganization of replication domains during embryonic stem cell differentiation.

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data.

BACKGROUND: Eukaryotic DNA replication is regulated at the level of large chromosomal domains (0.5-5 megabases in mammals) within which replicons are activated relatively synchronously. These domains replicate in a specific temporal order during S-phase and our genome-wide analyses of replication timing have demonstrated that this temporal order of domain replication is a stable property of specific cell types. RESULTS: We have developed ReplicationDomain http://www.replicationdomain.org as a web-based database for analysis of genome-wide replication timing maps (replication profiles) from various cell lines and species. This database also provides comparative information of transcriptional expression and is configured to display any genome-wide property (for instance, ChIP-Chip or ChIP-Seq data) via an interactive web interface. Our published microarray data sets are publicly available. Users may graphically display these data sets for a selected genomic region and download the data displayed as text files, or alternatively, download complete genome-wide data sets. Furthermore, we have implemented a user registration system that allows registered users to upload their own data sets. Upon uploading, registered users may choose to: (1) view their data sets privately without sharing; (2) share with other registered users; or (3) make their published or "in press" data sets publicly available, which can fulfill journal and funding agencies' requirements for data sharing. CONCLUSION: ReplicationDomain is a novel and powerful tool to facilitate the comparative visualization of replication timing in various cell types as well as other genome-wide chromatin features and is considerably faster and more convenient than existing browsers when viewing multi-megabase segments of chromosomes. Furthermore, the data upload function with the option of private viewing or sharing of data sets between registered users should be a valuable resource for the scientific community.

ANK1 Methylation regulates expression of MicroRNA-486-5p and discriminates lung tumors by histology and smoking status.

The intragenic tumor-suppressor microRNA miR-486-5p is often down-regulated in non-small cell lung cancer (NSCLC) but the mechanism is unclear. This study investigated epigenetic co-regulation of miR-486-5p and its host gene ANK1. MiR-486-5p expression in lung tumors and cell lines was significantly reduced compared to normal lung (p < 0.001) and is strongly correlated with ANK1 expression. In vitro, siRNA-mediated ANK1 knockdown in NSCLC cells also reduced miR-486-5p while the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced expression of both. ANK1 promoter CpG island was unmethylated in normal lung but methylated in 45% (118/262) lung tumors and 55% (17/31) NSCLC cell lines. After adjustment for tumor histology and smoking, methylation was significantly more prevalent in adenocarcinoma (101/200, 51%) compared to squamous cell carcinoma (17/62, 27%), p < 0.001; HR = 3.513 (CI: 1.818-6.788); and in smokers (73/128, 57%) than never-smokers (28/72, 39%), p = 0.014; HR = 2.086 (CI: 1.157-3.759). These results were independently validated using quantitative methylation data for 809 NSCLC cases from The Cancer Genome Atlas project. Together, our data indicate that aberrant ANK1 methylation is highly prevalent in lung cancer, discriminate tumors by histology and patients' smoking history, and contributes to miR-486-5p repression.

Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains.

BACKGROUND: Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains ('replication domains'), but few gene products have been identified whose disruption affects these properties. RESULTS: Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF. CONCLUSIONS: Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.

Developmental control of replication timing defines a new breed of chromosomal domains with a novel mechanism of chromatin unfolding.

We recently identified a set of chromosome domains that are early replicating uniquely in pluripotent cells. Their switch from early to late replication occurs just prior to germ layer commitment, associated with a stable form of gene silencing that is difficult to reverse. Here, we discuss results demonstrating that these domains are among the least sensitive regions in the genome to global digestion by either MNase or restriction enzymes. This inaccessible chromatin state persists whether these regions are in their physically distended early replicating or compact late replicating configuration, despite dramatic changes in 3D chromatin folding and long-range chromatin interactions, and despite large changes in transcriptional activity. This contrasts with the strong correlation between early replication, accessibility, transcriptional activity and open chromatin configuration that is observed genome-wide. We put these results in context with findings from other studies indicating that many structural (DNA sequence) and functional (density and activity of replication origins) properties of developmentally regulated replication timing ("switching") domains resemble properties of constitutively late replicating domains. This suggests that switching domains are a type of late replicating domain within which both replication timing and transcription are subject to unique or additional layers of control not experienced by the bulk of the genome. We predict that understanding the unusual structure of these domains will reveal a novel principle of chromosome folding.

Genome-scale analysis of replication timing: from bench to bioinformatics.

Replication timing profiles are cell type-specific and reflect genome organization changes during differentiation. In this protocol, we describe how to analyze genome-wide replication timing (RT) in mammalian cells. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) and sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated, amplified, differentially labeled and co-hybridized to a whole-genome comparative genomic hybridization microarray, which is currently more cost effective than high-throughput sequencing and equally capable of resolving features at the biologically relevant level of tens to hundreds of kilobases. We also present a guide to analyzing the resulting data sets based on methods we use routinely. Subjects include normalization, scaling and data quality measures, LOESS (local polynomial) smoothing of RT values, segmentation of data into domains and assignment of timing values to gene promoters. Finally, we cover clustering methods and means to relate changes in the replication program to gene expression and other genetic and epigenetic data sets. Some experience with R or similar programming languages is assumed. All together, the protocol takes ∼3 weeks per batch of samples.

DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.