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Emery-Dreifuss muscular dystrophy (EDMD) is a genetically and clinically variable disorder. Previous attempts to use gene expression changes find its pathomechanism were unavailing, so we here engaged a functional pathway analysis. RNA-Seq was performed on cells from 10 patients diagnosed with an EDMD spectrum disease with different mutations in 7 genes. Upon comparing to controls, the pathway analysis revealed that multiple genes involved in fibrosis, metabolism, myogenic signaling, and splicing were affected in all patients. Splice variant analysis revealed alterations of muscle-specific variants for several important muscle genes. Deeper analysis of metabolic pathways revealed a reduction in glycolytic and oxidative metabolism and reduced numbers of mitochondria across a larger set of 14 EDMD patients and 7 controls. Intriguingly, the gene expression signatures segregated the patients into three subgroups whose distinctions could potentially relate to differences in clinical presentation. Finally, differential expression analysis of miRNAs changing in the patients similarly highlighted fibrosis, metabolism, and myogenic signaling pathways. This pathway approach revealed a clear EDMD signature that can both be used as the basis for establishing a biomarker panel specific to EDMD and direct further investigation into its pathomechanism. Furthermore, the segregation of specific gene changes into three distinct categories that appear to correlate with clinical presentation may be developed into prognostic biomarkers, though this will first require their testing in a wider set of patients with more clinical information.

Background: As genome-wide approaches prove difficult with genetically heterogeneous orphan diseases, we developed a new approach to identify candidate genes. We applied this to Emery-Dreifuss muscular dystrophy (EDMD), characterised by early onset contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances with wide intra- and inter-familial clinical variability. Roughly half of EDMD patients are linked to six genes encoding nuclear envelope proteins, but the disease mechanism remains unclear because the affected proteins function in both cell mechanics and genome regulation. Methods: A primer library was generated to test for mutations in 301 genes from four categories: (I) all known EDMD-linked genes; (II) genes mutated in related muscular dystrophies; (III) candidates generated by exome sequencing in five families; (IV) functional candidates — other muscle nuclear envelope proteins functioning in mechanical/genome processes affected in EDMD. This was used to sequence 56 unlinked patients with EDMD-like phenotype. Findings: Twenty-one patients could be clearly assigned: 18 with mutations in genes of similar muscular dystrophies; 3 with previously missed mutations in EDMD-linked genes. The other categories yielded novel candidate genes, most encoding nuclear envelope proteins with functions in gene regulation. Interpretation: Our multi-pronged approach identified new disease alleles and many new candidate EDMD genes. Their known functions strongly argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction due to connectivity of candidates from the nuclear envelope to the plasma membrane. This approach highlights the value of testing for related diseases using primer libraries and may be applied for other genetically heterogeneous orphan diseases. Funding: The Wellcome Trust, Muscular Dystrophy UK, Medical Research Council, European Community's Seventh Framework Programme “Integrated European –omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)”. Keywords: Emery-Dreifuss muscular dystrophy, Nuclear envelope, Nuclear envelope transmembrane protein, primer library, Orphan disease

Limitations of genome-wide approaches for genetically-heterogenous orphan diseases led us to develop a new approach to identify novel Emery-Dreifuss muscular dystrophy (EDMD) candidate genes. We generated a primer library to genes: (I) linked to EDMD, (II) mutated in related muscular dystrophies, (III) highlighted from limited exome sequencing, (IV) encoding muscle-specific nuclear membrane proteins. Sequencing 56 unlinked EDMD patients yielded confirmed or strong candidate alleles from all categories, accounting for most remaining unlinked patients. Known functions of newly-linked genes argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction through connectivity of candidates from the nuclear envelope to the plasma membrane.

MBD1, a member of the methyl-CpG-binding domain family of proteins, has been reported to repress transcription of methylated and unmethylated promoters. As some MBD1 isoforms contain two DNA-binding domains-an MBD, which recognizes methylated DNA; and a CXXC3 zinc finger, which binds unmethylated CpG-it is unclear whether these two domains function independently of each other or if they cooperate in facilitating recruitment of MBD1 to particular genomic loci. In this report we investigate DNA-binding specificity of MBD and CXXC3 domains in vitro and in vivo. We find that the methyl-CpG-binding domain of MBD1 binds more efficiently to methylated DNA within a specific sequence context. We identify genes that are targeted by MBD1 in human cells and demonstrate that a functional MBD domain is necessary and sufficient for recruitment of MBD1 to specific sites at these loci, while DNA binding by the CXXC3 motif is largely dispensable. In summary, the binding preferences of MBD1, although dependent upon the presence of methylated DNA, are clearly distinct from those of other methyl-CpG-binding proteins, MBD2 and MeCP2.

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.

The cuckoo chromosome 4S(L) from Aegilops sharonensis is preferentially transmitted when introduced by hybridization into common wheat, Triticum aestivum. Gametocidal (Gc) factors carried in 4S(L) induce chromosome breakage in meiospores not containing them, ensuring their transmission to the progeny. Chromosome breakage and break-fusion-bridge (BFB) cycles can also be observed during early embryo sac development of chromosome 4S(L) addition lines to wheat, often leading to the presence of dicentric chromosomes in the subsequent progeny. However, the process responsible for inducing the primary chromosomal breaks only appears to occur during the initial divisions of the embryo and endosperm. In the presence of chromosome 4S(L), treatment with the hypomethylating agent 5-azacytidine induces chromosome breakage in root tips. This suggests that the process of chromosome fragmentation, induced by the Gc factors during early seed development, is repressed at later stages by DNA methylation.

LSH, a member of the SNF2 family of chromatin remodeling ATPases encoded by the Hells gene, is essential for normal levels of DNA methylation in the mammalian genome. While the role of LSH in the methylation of repetitive DNA sequences is well characterized, its contribution to the regulation of DNA methylation and the expression of protein-coding genes has not been studied in detail. In this report we investigate genome-wide patterns of DNA methylation at gene promoters in Hells−/− mouse embryonic fibroblasts (MEFs). We find that in the absence of LSH, DNA methylation is lost or significantly reduced at ∼20% of all normally methylated promoter sequences. As a consequence, a large number of genes are misexpressed in Hells−/− MEFs. Comparison of Hells−/− MEFs with wild-type MEFs and embryonic stem (ES) cells suggests that LSH is important for de novo DNA methylation events that accompany the establishment and differentiation of embryonic lineage cells. We further show that the generation of normal DNA methylation patterns and stable gene silencing at specific promoters require cooperation between LSH and the G9a/GLP complex of histone methylases. At such loci, G9a recruitment is compromised when LSH is absent or greatly reduced. Taken together, our data suggest a mechanism whereby LSH promotes binding of DNA methyltransferases and the G9a/GLP complex to specific loci and facilitates developmentally programmed DNA methylation and stable gene silencing during lineage commitment and differentiation.

Mammalian genome characterization and biotechnology each require the mobilization of large DNA segments to produce transgenic animals. We recently showed that mouse metaphase II (mII) oocytes could efficiently promote transgenesis (mII transgenesis) when coinjected with sperm and small (

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear how centromere sites are specified on chromosomes in vertebrates. We have previously introduced a mini-chromosome, named ST1, into a variety of cell lines including human HT1080, mouse LA9 and chicken DT40. This mini-chromosome, segregating faithfully in these cells, contains mouse minor and major, and human Y alpha-satellite DNA repeats. In this study, after determining the organisation of the satellite repeats, we investigated the location of the centromere on the mini-chromosome by combined immunocytochemistry and fluorescence in situ hybridisation analysis. Centromeric proteins were consistently co-localised with the minor satellite repeats in all three cell lines. When chromatin fibres were highly stretched, centromeric proteins were only seen on a small portion of the minor satellite repeats. These results indicate that a fraction of the minor satellite repeats is competent in centromere function not only in mouse but also in human and chicken cells.

We have developed a telomere-based episome by large-scale amplification in Escherichia coli cells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large-scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long-term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC-based episome is potentially a good candidate vector for gene therapy applications.

We describe a mammalian artificial mini-chromosome lacking human alphoid DNA and mouse minor and major satellite DNA repeats. This mini-chromosome, initially recovered in a mouse embryonic stem (ES) cell line (CGR8), is 2.6 Mb in size and consists of sequences derived from the human Y chromosome and mouse chromosomes 12 and 15. It is not stable in the CGR8 cells but replicates and segregates with high fidelity after transfer into chicken DT40 cells. Combined analysis by immunocytochemistry/fluorescence in situ hybridisation (FISH) on metaphase spreads detected an active neo-centromere on the mini-chromosome in these cells. Further analysis by immunocytochemistry/FISH on stretched chromatin allowed the localisation of the CENP-C protein to the DNA sequence derived from interval 5 of the human Y chromosome.