Heterochromatin Dynamics

Heterochromatin is usually thought of as a stable and inactive region of the genome. Not so, according to a study published earlier this yea

Differences in protein mobility between pioneer versus follower growth cones

Navigating growth cones need to integrate, process and respond to guidance signals, requiring dynamic information transfer within and between different compartments. Studies have shown that, faced with different navigation challenges, growth cones display dynamic changes in growth kinetics and morphologies. However, it remains unknown whether these are paralleled by differences in their internal molecular dynamics. To examine whether there are protein mobility differences during guidance, we developed multiphoton fluorescence recovery after photobleaching methods to determine molecular diffusion rates in pathfinding growth cones in vivo. Actively navigating growth cones (leaders) have consistently longer recovery times than growth cones that are fasciculated and less actively navigating (followers). Pharmacological perturbations of the cytoskeleton point to actin as the primary modulator of diffusion in differently behaving growth cones. This approach provides a powerful means to quantify mobility of specific proteins in neurons in vivo and reveals that diffusion is important during axon navigation

From the cradle to the grave:Green turtle hatchlings (Chelonia mydas) preyed upon by two-spots red snappers (Lutjanus bohar)

The observation of trophic interactions such as predation provide valuable information to model food webs and better understand ecosystem functioning. Such information is crucial for rare and endangered species in order to adapt management measures and ensure their conservation. However, trophic interactions are rarely observed in the marine realm, even for well-known or widespread species. During a scientific cruise in the Scattered Islands (Southwestern Indian Ocean), we observed endangered green turtle hatchlings (Chelonia mydas) in the gut content of two subadults two-spots red snappers (Lutjanus bohar). This trophic link involving emblematic species has not been previously described. The two-spots red snapper is a widespread coral reef fish in the tropical Indo-Pacific. Although it is unclear how fish predation affects marine turtle population dynamics, the occurrence of hatchlings in all the snapper samples suggests that fish could be significant sources of predation. Yet this predation pressure remains to be further studied and quantified to be considered in marine turtle population monitoring

Identification and characterization of a novel ubiquitous nucleolar protein ‘NARR’ encoded by a gene overlapping the rab34 oncogene

There are only few reports on protein products originating from overlapping mammalian genes even though computational predictions suggest that an appreciable fraction of mammalian genes could potentially overlap. Mass spectrometry-based proteomics has now acquired the tools to probe proteins in an unbiased manner, providing direct evidence of the output of the genomic and gene expression machinery. In particular, proteomics can refine gene predictions and discover novel gene-processing events and gene arrangements. Here, we report the mass spectrometric discovery and biochemical validation of the novel protein encoded by a gene overlapping rab34 oncogene. The novel protein is highly conserved in mammals. In humans, it contains 13 distinct Nine-Amino acid Residue-Repeats (NARR) with the consensus sequence PRVIV(S/T)PR in which the serine or threonine residues are phosphorylated during M-phase. NARR is ubiquitously expressed and resides in nucleoli where it colocalizes with ribosomal DNA (rDNA) gene clusters. Its distribution only partially overlaps with upstream binding factor, one of the main regulators of RNA Polymerase I activity, and is entirely uncoupled from it in mitotic cells and upon inhibition of transcription. NARR only partially colocalizes with fibrillarin, the pre-ribosomal RNA-processing protein, positioning NARR in a separate niche within the rDNA cluster

Intrabody-mediated diverting of HP1β to the cytoplasm induces co-aggregation of H3-H4 histones and lamin-B receptor

Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1β (HP1β) in living cells, we have generated a cytoplasmic targeted anti-HP1β intrabody, specifically directed against the C-terminal portion of the molecule. HP1β is a conserved component of mouse and human constitutive heterochromatin involved in diverse nuclear functions including gene silencing, DNA repair and nuclear membrane assembly. We found that the anti-HP1β intrabody sequesters HP1β into cytoplasmic aggregates, inhibiting its traffic to the nucleus. Lamin B receptor (LBR) and a subset of core histones (H3/H4) are also specifically co-sequestered in the cytoplasm of anti-HP1β intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1β intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1β:LBR containing aggregates. The expression of anti-HP1β scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1β or by HP1β mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1β-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1β. These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1β and its binding partners involved in peripheral heterochromatin organisation

Interplay of ribosomal DNA Loci in nucleolar dominance: dominant NORs are up-regulated by chromatin dynamics in the wheat-rye system

Background: Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. Methodology/Principal Findings: Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. Conclusions/Significance: We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are alsomodified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to ‘mutual amphiplasty’ in the nucleolar dominance process.This work was supported by the Fundação para a Ciência e Tecnologia (projects POCI/BIA-BDE/57575/2004 to M.S. and POCI/BIA-BCM/59389/2004 to N.N.

Heterochromatin protein 1 is recruited to various types of DNA damage

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage

Cbx4 maintains the epithelial lineage identity and cell proliferation in the developing stratified epithelium

During development, multipotent progenitor cells establish lineage-specific programmers of gene activation and silencing underlying their differentiation into specialized cell types. We show that the Polycomb component Cbx4 serves as a critical determinant that maintains the epithelial identity in the developing epidermis by repressing nonepidermal gene expression programs. Cbx4 ablation in mice results in a marked decrease of the epidermal thickness and keratinocyte (KC) proliferation associated with activation of numerous neuronal genes and genes encoding cyclin-dependent kinase inhibitors (p16/p19 and p57). Furthermore, the chromodomain- and SUMO E3 ligase–dependent Cbx4 activities differentially regulate proliferation, differentiation, and expression of nonepidermal genes in KCs. Finally, Cbx4 expression in KCs is directly regulated by p63 transcription factor, whereas Cbx4 overexpression is capable of partially rescuing the effects of p63 ablation on epidermal development. These data demonstrate that Cbx4 plays a crucial role in the p63-regulated program of epidermal differentiation, maintaining the epithelial identity and proliferative activity in KCs via repression of the selected nonepidermal lineage and cell cycle inhibitor genes

Nucleologenesis in the Caenorhabditis elegans Embryo

In the Caenorhabditis elegans nematode, the oocyte nucleolus disappears prior to fertilization. We have now investigated the re-formation of the nucleolus in the early embryo of this model organism by immunostaining for fibrillarin and DAO-5, a putative NOLC1/Nopp140 homolog involved in ribosome assembly. We find that labeled nucleoli first appear in somatic cells at around the 8-cell stage, at a time when transcription of the embryonic genome begins. Quantitative analysis of radial positioning showed the nucleolus to be localized at the nuclear periphery in a majority of early embryonic nuclei. At the ultrastructural level, the embryonic nucleolus appears to be composed of a relatively homogenous core surrounded by a crescent-shaped granular structure. Prior to embryonic genome activation, fibrillarin and DAO-5 staining is seen in numerous small nucleoplasmic foci. This staining pattern persists in the germline up to the ∼100-cell stage, until the P4 germ cell divides to give rise to the Z2/Z3 primordial germ cells and embryonic transcription is activated in this lineage. In the ncl-1 mutant, which is characterized by increased transcription of rDNA, DAO-5-labeled nucleoli are already present at the 2-cell stage. Our results suggest a link between the activation of transcription and the initial formation of nucleoli in the C. elegans embryo