Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors

Emerin Caps the Pointed End of Actin Filaments: Evidence for an Actin Cortical Network at the Nuclear Inner Membrane

X-linked Emery-Dreifuss muscular dystrophy is caused by loss of emerin, a LEM-domain protein of the nuclear inner membrane. To better understand emerin function, we used affinity chromatography to purify emerin-binding proteins from nuclear extracts of HeLa cells. Complexes that included actin, αII-spectrin and additional proteins, bound specifically to emerin. Actin polymerization assays in the presence or absence of gelsolin or capping protein showed that emerin binds and stabilizes the pointed end of actin filaments, increasing the actin polymerization rate 4- to 12-fold. We propose that emerin contributes to the formation of an actin-based cortical network at the nuclear inner membrane, conceptually analogous to the actin cortical network at the plasma membrane. Thus, in addition to disrupting transcription factors that bind emerin, loss of emerin may destabilize nuclear envelope architecture by weakening a nuclear actin network

Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL

The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion

Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly: Biochemical Demonstration and Molecular Analysis

The nuclear pore complex (NPC) is characterized by a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel, within which the nuclear pore is built, has little evolutionary precedent. In this report we demonstrate and map the inner/outer nuclear membrane fusion in NPC assembly

Tracking Endogenous Amelogenin and Ameloblastin In Vivo

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling

Barrier-to-autointegration factor is required to segregate and enclose chromosomes within the nuclear envelope and assemble the nuclear lamina

Barrier-to-autointegration factor (BAF) binds dsDNA, LEM-domain proteins, and lamins. Caenorhabditis elegans BAF requires Ce-lamin and two LEM-domain proteins (Ce-emerin and Ce-MAN1) to localize during nuclear assembly. It was unknown whether Ce-lamin and LEM proteins, in turn, depend on Ce-BAF (mutually dependent structural roles). RNA interference-mediated down-regulation of Ce-BAF caused gross defects in chromosome segregation, chromatin decondensation, and mitotic progression as early as the two-cell stage, and embryos died at the ≈100-cell stage. Nuclear pores reassembled, whereas Ce-lamin, Ce-emerin, and Ce-MAN1 bound chromatin but remained patchy and disorganized. The nuclear membranes formed but failed to enclose anaphase-bridged chromatin. Time-lapse imaging showed two phenotypes: anaphase-bridged chromatin that eventually resolved, and segregated chromatin that returned to the midzone. Thus, the assembly of BAF, lamins, and LEM-domain proteins is mutually dependent, and is required to capture segregated chromosomes within the nascent nuclear envelope. Embryos that escaped lethality by down-regulation of Ce-BAF grew into sterile adults with misplaced distal tip cells and gonads, further suggesting that mild postembryonic reductions in BAF disrupt tissue-specific functions