Prdm16-mediated H3K9 methylation controls fibro-adipogenic progenitors identity during skeletal muscle repair.

H3K9 methylation maintains cell identity orchestrating stable silencing and anchoring of alternate fate genes within the heterochromatic compartment underneath the nuclear lamina (NL). However, how cell type-specific genomic regions are specifically targeted to the NL is still elusive. Using fibro-adipogenic progenitors (FAPs) as a model, we identified Prdm16 as a nuclear envelope protein that anchors H3K9-methylated chromatin in a cell-specific manner. We show that Prdm16 mediates FAP developmental capacities by orchestrating lamina-associated domain organization and heterochromatin sequestration at the nuclear periphery. We found that Prdm16 localizes at the NL where it cooperates with the H3K9 methyltransferases G9a/GLP to mediate tethering and silencing of myogenic genes, thus repressing an alternative myogenic fate in FAPs. Genetic and pharmacological disruption of this repressive pathway confers to FAP myogenic competence, preventing fibro-adipogenic degeneration of dystrophic muscles. In summary, we reveal a druggable mechanism of heterochromatin perinuclear sequestration exploitable to reprogram FAPs in vivo.

SAMMY-seq reveals early alteration of heterochromatin and deregulation of bivalent genes in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome is a genetic disease caused by an aberrant form of Lamin A resulting in chromatin structure disruption, in particular by interfering with lamina associated domains. Early molecular alterations involved in chromatin remodeling have not been identified thus far. Here, we present SAMMY-seq, a high-throughput sequencing-based method for genome-wide characterization of heterochromatin dynamics. Using SAMMY-seq, we detect early stage alterations of heterochromatin structure in progeria primary fibroblasts. These structural changes do not disrupt the distribution of H3K9me3 in early passage cells, thus suggesting that chromatin rearrangements precede H3K9me3 alterations described at later passages. On the other hand, we observe an interplay between changes in chromatin accessibility and Polycomb regulation, with site-specific H3K27me3 variations and transcriptional dysregulation of bivalent genes. We conclude that the correct assembly of lamina associated domains is functionally connected to the Polycomb repression and rapidly lost in early molecular events of progeria pathogenesis.

Determination of Polycomb Group of Protein Compartmentalization Through Chromatin Fractionation Procedure.

Epigenetic mechanisms modulate and maintain the transcriptional state of the genome acting at various levels on chromatin. Emerging findings suggest that the position in the nuclear space and the cross talk between components of the nuclear architecture play a role in the regulation of epigenetic signatures. We recently described a cross talk between the Polycomb group of proteins (PcG) epigenetic repressors and the nuclear lamina. This interplay is important for the maintenance of transcriptional repression at muscle-specific genes and for the correct timing of muscle differentiation. To investigate the synergism between PcG factors and nuclear architecture we improved a chromatin fractionation protocol with the aim to analyze the PcG nuclear compartmentalization. We thus separated PcG proteins in different fractions depending on their solubility. We surprisingly found a consistent amount of PcG proteins in the matrix-associated fraction. In this chapter we describe the chromatin fractionation procedure, a method that can be used to study the nuclear compartmentalization of Polycomb group of proteins and/or PcG targets in murine and Drosophila cells.

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.

Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress.

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G 1- or G 2-specific differentiation checkpoint. In response to genotoxins that induce G 1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G 2 checkpoint relies on the inability of MyoD to bind the chromatin at the G 2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.

An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis.

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

Phosphodiesterase type IV inhibition prevents sequestration of CREB binding protein, protects striatal parvalbumin interneurons and rescues motor deficits in the R6/2 mouse model of Huntington's disease.

The phosphodiesterase type IV inhibitor rolipram increases cAMP response element-binding protein (CREB) phosphorylation and exerts neuroprotective effects in both the quinolinic acid rat model of Huntington's disease (DeMarch et al., 2007) and the R6/2 mouse including sparing of striatal neurons, prevention of neuronal intranuclear inclusion formation and attenuation of microglial reaction (DeMarch et al., 2008). In this study, we sought to determine if rolipram has a beneficial role in the altered distribution of CREB binding protein in striatal spiny neurons and in the motor impairments shown by R6/2 mutants. Moreover, we investigated whether rolipram treatment altered the degeneration of parvalbuminergic interneurons typical of Huntington's disease (Fusco et al., 1999). Transgenic mice and their wild-type controls from a stable colony maintained in our laboratory were treated with rolipram (1.5 mg/kg) or saline daily starting from 4 weeks of age. The cellular distribution of CREB binding protein in striatal spiny neurons was assessed by immunofluorescence, whereas parvalbuminergic neuron degeneration was evaluated by cell counts of immunohistochemically labeled tissue. Motor coordination and motor activity were also examined. We found that rolipram was effective in preventing CREB binding protein sequestration into striatal neuronal intranuclear inclusions, sparing parvalbuminergic interneurons of R6/2 mice, and rescuing their motor coordination and motor activity deficits. Our findings demonstrate the possibility of reversing pharmacologically the behavioral and neuropathological abnormalities of symptomatic R6/2 mice and underline the potential therapeutic value of phosphodiesterase type IV inhibitors in Huntington's disease.

Phosphodiesterase 10 inhibition reduces striatal excitotoxicity in the quinolinic acid model of Huntington's disease.

Decreased activity of cAMP responsive element-binding protein (CREB) is thought to contribute to the death of striatal medium spiny neurons in Huntington's disease (HD). Therefore, therapies that increase levels of activated CREB, may be effective in fighting neurodegeneration in HD. In this study, we sought to determine whether the phosphodiesterase type 10 (PDE10A) inhibitor TP10 exerts a neuroprotective effect in an excitotoxic model of HD. Rats were surgically administered with quinolinic acid into striatum and subsequently treated with TP10 daily for two or eight weeks. After 2 weeks of TP10 treatment, striatal lesion size was 52% smaller and the surviving cell number was several times higher than in the vehicle-treated group. These beneficial effects of TP10 were maintained through 8 weeks. TP10 treatment also increased significantly the levels of activated CREB in the striatal spiny neurons, which is hypothesized to be a contributing mechanism for the neuroprotective effect. Our findings suggest PDE10A inhibition as a novel neuroprotective approach to the treatment of HD and confirm the importance of phosphodiesterase inhibition in fighting the disease.