Role of MeCP2 (Meyhyl CpG-Binding Protein) during replication, epigenetic inheritance and chromocenter organization of pericentric heterochromatin.

\u201cEpigenetic\u201d is a term used to describe mitotically and meiotically heritable states of gene expression that are not due to changes in DNA sequence. Epigenetic events are important in all aspects of biology such as cell proliferation, development and differentiation. The maintenance of a correct epigenome is fundamental in the proper progression of silencing effect, pivotal during cellular differentiation. Multi-protein complexes required to enable the heterochromatic stable epigenetic inheritance are associated to the structural organization of heterochromatin that is determined at the time of replication, in mid-late S phase. MeCP2 is a Methyl CpG-binding protein that preferentially binds methylated DNA, localizes with pericentric heterochromatin and it has a key role to mediate large-scale chromatin organization and compaction. While the impact of loss and mutations of MeCP2 has been extensively studied as cause of RTT and other neurodevelopmental and autismspectrum diseases little is known about its function in pericentric heterochromatin replication, structure and epigenetic inheritance. To test whether MeCP2 has a role in these processes, I used proliferating cells to study the effect of MeCP2 functional ablation during cell-cycle S-phase. I found that MeCP2 is not involved in heterochromatin replication, chromocenter organization and epigenetic modifications (H3K9me3, H4K20me3 and DNA methylation). Interestingly, MeCP2 influences cell cycle progression without triggering a strong apoptotic effect. Intriguingly, low levels of LaminB, LBR and LaminA/C proteins were found, suggesting that MeCP2 could be involved in nuclear lamina organization and/or in the expression of these genes. Besides, MeCP2 silencing determines low levels of EZH2 (component of PRC2, one of the two Polycomb-Repressive Complexes). In the light of this finding is intriguing to investigate the link between MeCP2 and EZH2 due the fact that a large number of neuronal differentiation genes which are required for neuronal development cells are bound by Polycomb complexes, whereas MeCP2 is a transcriptional regulator implicated in development of the brain that is required to interpret the DNA methylation signal in neurons. In parallel, using different biochemical tools (TAP, gel filtration, mass spectrometry) I investigated the ability of MeCP2 to bind proteins involved in heterochromatin organization. A new interactor Np95, component of pHDBs (pericentric heterochromatin duplication bodies), was found. My results show that MeCP2 in addition to play a role in maturation of neurons and synaptic plasticity it is implicated in cell proliferation. Besides, MeCP2 might be involved in nuclear envelope stability and/or in the expression of lamins, and in Polycomb proteins pathway. On the basis of this work further experiments should be done to better investigate the new functions of MeCP2

UHRF1-miRNAs modulate Dnmt3a expression in prostate transformed cells

UHRF1 is overexpressed in many tumours and is an epigenetic regulator required for DNA methylation (DNAm) by recruiting all DNMTs to methylation sites. UHRF1 is also crucial for histone modifications, epigenetic gene silencing and has an important impact on prostate cancer pathogenesis and progression. Here we studied whether UHRF1 could exert its role in the epigenetic of cancer by changes in microRNA (miRNA) levels in prostate transformed cells. MiRNAs are small non-coding RNAs that negatively control gene expression and play crucial functions in all cellular processes. Growing evidences indicate their deregulated expression in human cancer, proposing miRNAs as oncogenes or tumour suppressors. Besides, miRNAs can induce gene silencing via epigenetic mechanisms e.g. by targeting a specific gene region for DNAm and histone modifications or by regulating expression of epigenetic enzymes. MiRNAs expression profile of siRNA-UHRF1 PC3 cells showed both increased and decreased miRNA levels. Using different target predictive software, Dnmt3a appeared as a target of two new miRNAs (miRNA-a and miRNA-b) overexpressed in siRNA-UHRF1 PC3 cells. Levels of miRNA-a and -b were confirmed by RT-QPCR in siRNA-UHRF1 PC3 and in LNCaP cells (with UHRF1 and Dnmt3a lower levels than PC3) and the binding of the two new miRNAs to the Dnmt3a3\u2019UTR and its inhibitory effect were validated by reporter luciferase system. We studied the effect of the overexpression of miRNA-a and -b in PC3 finding a decrease of Dnmt3a mRNA and protein, and a decrease in PC3 proliferation. We studied the effects of miRNA silencing in LNCaP finding an increase of Dnmt3a mRNA and protein, and an increase in LNCaP proliferation. These data show the role of miRNA-a and -b in the modulation of Dnmt3a and propose their involvement in prostate cancer progression. Next goal is to study their promoters evaluating the role of UHRF1 in miRNAs transcription

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

Poly(ADP-ribose) polymerase 1 (PARP1) associates with E3 ubiquitin-protein ligase UHRF1 and modulates UHRF1 biological functions

Poly(ADP-ribose) polymerase 1 (PARP1, also known as ARTD1) is an abundant nuclear enzyme that plays important roles in DNA repair, gene transcription, and differentiation through the modulation of chromatin structure and function. In this work we identify a physical and functional poly(ADP-ribose)-mediated interaction of PARP1 with the E3 ubiquitin ligase UHRF1 (also known as NP95, ICBP90) that influences two UHRF1-regulated cellular processes. On the one hand, we uncovered a cooperative interplay between PARP1 and UHRF1 in the accumulation of the heterochromatin repressive mark H4K20me3. The absence of PARP1 led to reduced accumulation of H4K20me3 onto pericentric heterochromatin that coincided with abnormally enhanced transcription. The loss of H4K20me3 was rescued by the additional depletion of UHRF1. In contrast, although PARP1 also seemed to facilitate the association of UHRF1 with DNMT1, its absence did not impair the loading of DNMT1 onto heterochromatin or the methylation of pericentric regions, possibly owing to a compensating interaction of DNMT1 with PCNA. On the other hand, we showed that PARP1 controls the UHRF1-mediated ubiquitination of DNMT1 to timely regulate its abundance during S and G2 phase. Together, this report identifies PARP1 as a novel modulator of two UHRF1-regulated heterochromatin-associated events: the accumulation of H4K20me3 and the clearance of DNMT1

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression

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

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