Formation of nuclear heterochromatin: the nucleolar point of view.

Establishment and inheritance of heterochromatic states is critical in maintaining genome integrity and gene expression state. The elucidation of the mechanisms implicated in these processes is fundamental to understand the control of epigenetic regulation of the genome. Recently, the nucleolus emerged as an important component of the nuclear architecture. Although the nucleolus is the most active site of cellular transcription, it is also an attractive compartment for nuclear heterochromatic regions, such as pericentric repeats, inactive X chromosome and regions with low gene density significantly enriched in repressed genes. The coexistence of euchromatic and heterochromatic rRNA genes in each cell reflects these two opposite functions of the nucleolus. An epigenetic network that is controlled by NoRC complex establishes and maintains rDNA heterochromatin. It is here discussed how heterochromatic rRNA genes and the associated epigenetic regulatory activities might mediate formation and inheritance of nuclear heterochromatic regions. Finally, we propose that the analysis of the components of heterochromatic rRNA genes will be not only relevant to understand the general composition of heterochromatin but has the potential to provide important and novel insights of how nuclear heterochromatic structures are established and inherited.

Inheritance of silent rDNA chromatin is mediated by PARP1 via noncoding RNA.

Faithful propagation of specific chromatin states requires re-establishment of epigenetic marks after every cell division. How the original epigenetic signature is inherited after disruption during DNA replication is still poorly understood. Here, we show that the poly(ADP-ribose)-polymerase-1 (PARP1/ARTD1) is implicated in the maintenance of silent rDNA chromatin during cell division. We demonstrate that PARP1 associates with TIP5, a subunit of the NoRC complex, via the noncoding pRNA and binds to silent rRNA genes after their replication in mid-late S phase. PARP1 represses rRNA transcription and is implicated in the formation of silent rDNA chromatin. Silent rDNA chromatin is a specific substrate for ADP-ribosylation and the enzymatic activity of PARP1 is necessary to establish rDNA silencing. The data unravel a function of PARP1 and ADP-ribosylation that serves to allow for the inheritance of silent chromatin structures, shedding light on how epigenetic marks are transmitted during each cell cycle.

Munc18b regulates core SNARE complex assembly and constitutive exocytosis by interacting with the N-peptide and the closed-conformation C-terminus of syntaxin 3.

The interaction between SM (Sec1/Munc18) and SNARE (soluble N-ethylmaleimide-sensitive factor-attachment receptor) proteins constitutes the core eukaryotic membrane fusion machinery which manages exocytosis by mediating fusion of constitutively exocytic vesicles with the plasma membrane. However, mechanistic details on the nature and the physiological impact of SM-SNARE interactions remain largely elusive. Detailed characterization of the interaction profiles between Munc18b and its cognate SNAREs, Stx3 (syntaxin 3), SNAP-23 (soluble N-ethylmaleimide-attachment protein 23) and VAMP8 (vesicle-associated membrane protein 8), revealed that Munc18b binds Stx3, VAMP8 and the assembled core SNARE complex consisting of Stx3, SNAP-23 and VAMP8. Dissection of the Munc18b-Stx3 heterodimer suggested that Munc18b interacts with Stx3's conserved N-peptide as well as with its closed-conformation C-terminus encompassing the Habc domain, a linker and the SNARE (H3) motif. Deletion of the Habc domain or mutations interrupting the intramolecular binding of the Habc and H3 domains abrogated the Munc18b-Stx3 interaction. Although only the N-peptide deletion mutant, but not the soluble wild-type Stx3, is assembled into the core SNARE complex in the presence of Munc18b in vitro, ectopic expression of this SM protein increases constitutive exocytosis in mammalian cells. Our results suggest that Munc18b is functionally coupled to the assembly of exocytic SNARE complexes and increases exocytosis by interacting with the N-peptide and closed-conformation C-terminus of Stx3, thereby neutralizing the secretion-inhibitory effect of this SNARE.

The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats.

Maintenance of specific heterochromatic domains is crucial for genome stability. In eukaryotic cells, a fraction of the tandem-repeated ribosomal RNA (rRNA) genes is organized in the heterochromatic structures. The principal determinant of rDNA silencing is the nucleolar remodelling complex, NoRC, that consists of TIP5 (TTF-1-interacting protein-5) and the ATPase SNF2h. Here we showed that TIP5 not only mediates the establishment of rDNA silencing but also the formation of perinucleolar heterochromatin that contains centric and pericentric repeats. Our data indicated that the TIP5-mediated heterochromatin is indispensable for stability of silent rRNA genes and of major and minor satellite repeats. Moreover, depletion of TIP5 impairs rDNA silencing, upregulates rDNA transcription levels and induces cell transformation. These findings point to a role of TIP5 in protecting genome stability and suggest that it can play a role in the cellular transformation process.

The vesicle-trafficking protein munc18b increases the secretory capacity of mammalian cells.

Heterologous protein production in mammalian cells is often challenged by the bottleneck of the secretory machinery, which prevents producer cells from fully exploiting their physiologic capacity in the production of biopharmaceuticals. Recent advances in the understanding of the molecular mechanisms of vesicle trafficking have enabled the identification of key regulators that control the flow of recombinant proteins along the secretory pathway. Here, we report that transgenic expression of Munc18b, a Sec1/Munc18 (SM) protein regulating the fusion of secretory vesicles to the plasma membrane, enhances the secretory capacity of HeLa, HEK-293 and HT-1080 and so increases overall production of different secreted human glycoproteins as well as the titer of lentiviral particles produced in HEK-293-derived helper cells. Targeted interventions in secretory vesicle trafficking by Munc18b is a novel secretion engineering strategy, which harnesses the full secretory capacity of mammalian cells. Secretion engineering is the latest-generation metabolic engineering strategy, which could improve future therapies by increasing the production of biopharmaceuticals by boosting the secretion performance of cell implants in cell therapy initiatives and by raising the production titers of transgenic viral particles used for gene therapy applications.