Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations

• Main Author: Perovanovic, Jelena
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2015
• Subjects: Bioinformatics; Developmental biology; Genetics
• ISBN: 1321922574; 9781321922578

The nuclear envelope is important to myogenesis, where the transition of lamin B to lamin A/C during terminal myogenic differentiation is needed for correct muscle development. Gene mutations of inner nuclear envelope components (LMNA, EDM) cause muscular dystrophy phenotypes (Emery-Dreifuss muscular dystrophy [EDMD]; limb-girdle muscular dystrophy). There is considerable allelic and clinical heterogeneity of the laminopathies, where distinct LMNA mutations selectively affect striated muscle, adipose, peripheral nerve, or multiple systems. These nuclear envelope components provide structural support to eukaryotic nuclei, but emerging data suggest key roles in DNA replication, transcription and chromatin organization. Previous studies in our lab have shown that patients with EDMD show specific alterations of MyoD/Rb pathways compared to other dystrophies (Bakay et al., 2006), and further that emerin null mice show a specific block in myogenesis during terminal differentiation of myoblasts (Melcon et al., 2006). In this dissertation it is shown that mutations in LMNA and loss of emerin causing EDMD lead to disruption of developmentally appropriate chromatin-nuclear envelope associations during myogenesis. We scanned for genomic loci interacting with lamin A protein during myogenic differentiation using DamID-seq, and determined the effect of disease-associated amino acid changes on these protein/chromatin interactions (p.R453W Emery-Dreifuss Muscular Dystrophy; p.R482W Familial Partial Lipodystrophy). Lamin A missense mutations caused both promiscuous formation of lamin A associated heterochromatin domains (LAD), and decreased the length of WT LADs. These epigenetic alterations corresponded with pervasive presence of lamin A p.R452W (EDMD) at myogenic loci that was not seen with WT and p.R482W (FPLD). This finding was validated by DNA methylation studies in EDMD patient myogenic cells (WT vs. p.H222P). Also, appropriate exit from the cell cycle was perturbed by both mutations, with CDK1 and RB1 loci showing failure to attain LAD-mediated heterochromatin formation by DamID-seq and Chromatin Immuprecipitation sequencing ChIP-seq (H3K9me3 heterochromatin mark) analyses. Epigenetic findings in EDMD-AD (lamin A missense) were shared with the clinical phenocopy EDMD-XR (lamin A-associated emerin loss of function), with ChIP-seq of emerin null myogenic cells showing loss of heterochromatin marks at Sox2 and pluripotency pathway members. The failure of epigenetic remodeling at Sox2 was validated by mRNA (in vitro, and in EDMD patient muscle biopsies), and by lamin A p.H222P ChIP-qPCR. The alterations of SOX2 epigenetics may be upstream of the observed myogenic and cell cycle changes, as over-expression of SOX2 inhibited myogenic differentiation in vitro. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming caused by altered chromatin tethering to the nuclear periphery.