Np95 è coinvolta nel mantenimento dell’informazione epigenetica della cromatina associata a repressione trascrizionale in cellule replicanti e P19 indotte a differenziare a neuroni.

missin

Np95 è coinvolta nel mantenimento dell’informazione epigenetica della cromatina associata a repressione trascrizionale in cellule replicanti e P19 indotte a differenziare a neuroni.

missin

Abstract 196: UHRF1 is upregulated in prostate cancer and induces epigenetic silencing of tumor suppressor genes

Cancer of the prostate is the most common cancer and a leading cause of cancer death in Europe and North America. At the present there is a need to understand the mechanisms involved in the pathogenesis of this disease and discover alternative therapeutic targets. UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, has been previously reported to acts as a dominant negative effectors of cell growth. UHRF1 is involved in epigenetic mechanisms by virtue of its interaction with DNMTs and HMTs. In this study, analysis of gene expression profiles of prostate tumors and normal prostate showed that UHRF1 is frequently over-expressed in tumors compared to normal tissues. UHRF1 expression was very low in immortalized prostate epithelial cells (LH) while it was higher in the Ras transformed counterpart LHSR cells. Furthermore, we observed low level of UHRF1 in the androgen-dependent prostate cancer cell lines LNCaP and 22Rv1, while higher levels were present in the androgen-independent cell lines PC3 and DU145. These data suggested that over-expression of UHRF1 is associated with malignant transformation and prostate cancer progression. To understand the role of UHRF1 we performed knockdown experiments with UHRF1 specific siRNA. Transient transfection in PC3 cells reduced UHRF1 mRNA and protein level. UHRF1 knock-down resulted in reversion of the transformed phenotype with significant inhibition of clonogenic growth in anchorage dependent and independent condition. Reversion of the transformed phenotype occurred concomitantly with restoration of the expression of several tumor suppressor genes relevant for prostate differentiation, proliferation and epithelial-mesenchymal transition such as CDH1 and RARB2. Moreover, chromatin immunoprecipitation showed binding of UHRF1 to these gene promoter and UHRF1 knockdown resulted in significant reduction of repressive histone marks on gene promoters. These studies suggest that deregulated UHRF1 expression results in epigenetic silencing of relevant tumor suppressor genes, contributing to prostate cancer progression. Targeting UHRF1 may result in re-expression of tumor suppressor genes and thus may be a valid strategy for therapeutic intervention

Ultrafast response of harmonic modelocked THz lasers

International audienceThe use of fundamental modelocking to generate short terahertz (THz) pulses and THz frequency combs from semiconductor lasers has become a routine affair, using quantum cascade lasers (QCLs) as a gain medium. However, unlike classic laser diodes, no demonstrations of harmonic modelocking, active or passive, have been shown in THz QCLs, where multiple pulses per round trip are generated when the laser is modulated at the harmonics of the cavity's fundamental round-trip frequency. Here, using time-resolved THz techniques, we show for the first time harmonic injection and mode-locking in which THz QCLs are modulated at the harmonics of the round-trip frequency. We demonstrate the generation of the harmonic electrical beatnote within a QCL, its injection locking to an active modulation and its direct translation to harmonic pulse generation using the unique ultrafast nature of our approach. Finally, we show indications of self-starting harmonic emission, i.e., without external modulation, where the QCL operates exclusively on a harmonic (up to its 15th harmonic) of the round-trip frequency. This behaviour is supported by time-resolved simulations of induced gain and loss in the system and shows the importance of the electronic, as well as photonic, nature of QCLs. These results open up the prospect of passive harmonic modelocking and THz pulse generation, as well as the generation of low-noise microwave generation in the hundreds of GHz region

DNMT3A epigenetically regulates key microRNAs involved in epithelial-to-mesenchymal transition in prostate cancer

: Epithelial-to-Mesenchymal Transition (EMT) is involved in prostate cancer metastatic progression, and its plasticity suggests epigenetic implications. Deregulation of DNMTs and several miRNAs plays a relevant role in EMT, but their interplay has not been clarified yet. In this study we provide evidence that DNMT3A interaction with several miRNAs has a central role in an ex-vivo EMT prostate cancer model obtained via exposure of PC3 cells to conditioned media from cancer-associated fibroblasts (CM-CAFs). The analysis of the alterations of the miRNA profile shows that miR-200 family (miR-200a/200b/429, miR-200c/141), miR-205, and miR-203, known to modulate key EMT factors, are downregulated and hyper-methylated at their promoters. DNMT3A (mainly isoform a) is recruited onto these miRNA promoters, coupled with the increase of H3K27me3/H3K9me3 and/or the decrease of H3K4me3/H3K36me3. Most interestingly, our results reveal the differential expression of two DNMT3A isoforms (a and b) during ex-vivo EMT and a regulatory feedback loop between miR-429 and DNMT3A that can promote and sustain the transition toward a more mesenchymal phenotype. We demonstrate the ability of miR-429 to target DNMT3A 3'UTR and modulate the expression of EMT factors, in particular ZEB1. Survey of the PRAD-TCGA data set shows that patients expressing an EMT-like signature are indeed characterized by down-regulation of the same miRNAs with a diffused hyper-methylation at miR-200c/141 and miR-200a/200b/429 promoters. Finally, we show that miR-1260a also targets DNMT3A, although it does not seem involved in EMT in prostate cancer

Np95 Is Implicated in Pericentromeric Heterochromatin Replication and in Major Satellite Silencing

Heterochromatin plays an important role in transcriptional repression, for the correct segregation of chromosomes and in the maintenance of genome stability. Pericentric heterochromatin (PH) replication and formation have been proposed to occur in the pericentric heterochromatin duplication body (pHDB). A central question is how the underacetylated state of heterochromatic histone H4 tail is established and controlled, because it is a key event during PH replication and is essential to maintain the compacted and silenced state of these regions. Np95 is a cell cycle regulated and is a nuclear histone-binding protein that also recruits HDAC-1 to target promoters. It is essential for S phase and for embryonic formation and is implicated in chromosome stability. Here we show that Np95 is part of the pHDB, and its functional ablation causes a strong reduction in PH replication. Depletion of Np95 also causes a hyperacetylation of lysines 8, 12, and 16 of heterochromatin histone H4 and an increase of pericentromeric major satellite transcription, whose RNAs are key players for heterochromatin formation. We propose that Np95 is a new relevant protein involved in heterochromatin replication and formation

Knock-down of methyl CpG-binding protein 2 (MeCP2) causes alterations in cell proliferation and nuclear lamins expression in mammalian cells

<p>Abstract</p> <p>Background</p> <p>MeCP2 (CpG-binding protein 2) is a nuclear multifunctional protein involved in several cellular processes, like large-scale chromatin reorganization and architecture, and transcriptional regulation. In recent years, a non-neuronal role for MeCP2 has emerged in cell growth and proliferation. Mutations in the MeCP2 gene have been reported to determine growth disadvantages in cultured lymphocyte cells, and its functional ablation suppresses cell growth in glial cells and proliferation in mesenchymal stem cells and prostate cancer cells. MeCP2 interacts with lamin B receptor (LBR) and with Heterochromatin Protein 1 (HP1) at the nuclear envelope (NE), suggesting that it could be part of complexes involved in attracting heterochromatin at the nuclear periphery and in mediating gene silencing. The nuclear lamins, major components of the lamina, have a role in maintaining NE integrity, in orchestrating mitosis, in DNA replication and transcription, in regulation of mitosis and apoptosis and in providing anchoring sites for chromatin domains.</p> <p>In this work, we inferred that MeCP2 might have a role in nuclear envelope stability, thereby affecting the proliferation pattern of highly proliferating systems.</p> <p>Results</p> <p>By performing knock-down (KD) of MeCP2 in normal murine (NIH-3 T3) and in human prostate transformed cells (PC-3 and LNCaP), we observed a strong proliferation decrease and a defect in the cell cycle progression, with accumulation of cells in S/G₂M, without triggering a strong apoptotic and senescent phenotype. In these cells, KD of MeCP2 evidenced a considerable decrease of the levels of lamin A, lamin C, lamin B1 and LBR proteins. Moreover, by confocal analysis we confirmed the reduction of lamin A levels, but we also observed an alteration in the shape of the nuclear lamina and an irregular nuclear rim.</p> <p>Conclusions</p> <p>Our results that indicate reduced levels of NE components, are consistent with a hypothesis that the deficiency of MeCP2 might cause the lack of a key “bridge” function that links the peripheral heterochromatin to the NE, thereby causing an incorrect assembly of the NE itself, together with a decreased cell proliferation and viability.</p

The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH