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\u201320 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

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

Expression and localization studies of hSDA, the human ortholog of the yeast SDA1 gene

The yeast SDA1 gene was reported to play a critical role in G 1 events and to be involved in 60S ribosome biogenesis. Although the basic cellular mechanisms appear conserved from yeast to man, the human genes may have more diversified functions. In this view we obtained the first experimental evidences about the human ortholog of the yeast SDA1, i.e., hSDA. The gene is localized at the chromosomal region 4q21 and encodes for a 627a.a. long protein highly homologous to the yeast Sda1. Subcellular localization experiments indicate that the human protein behaves similarly to nucleolar proteins involved in rRNA processing machinery but not in RNA Poll transcriptional events. hSda appears localized in the granular component of the nucleolus and in the nucleoplasm, which is consistent with a role in early-intermediate steps of ribosome biogenesis. hSDA appears preferentially expressed in fetal tissues, pinpointing its role during development. Different expression levels in different tumor cell lines might suggest that the gene is involved also in tumorigenesis. However our preliminary results indicate that hSDA does not behave like a proapoptotic gene and its involvement in tumorigenesis is still to be clarified

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

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

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

UHRF1 coordinates peroxisome proliferator activated receptor gamma (PPARG) epigenetic silencing and mediates colorectal cancer progression.

Peroxisome proliferator-activated receptor gamma (PPARG) inactivation has been identified as an important step in colorectal cancer (CRC) progression, although the events involved have been partially clarified. UHRF1 is emerging as a cofactor that coordinates the epigenetic silencing of tumor suppressor genes, but its role in CRC remains elusive. Here, we report that UHRF1 negatively regulates PPARG and is associated with a higher proliferative, clonogenic and migration potential. Consistently, UHRF1 ectopic expression induces PPARG repression through its recruitment on the PPARG promoter fostering DNA methylation and histone repressive modifications. In agreement, UHRF1 knockdown elicits PPARG re-activation, accompanied by positive histone marks and DNA demethylation, corroborating its role in PPARG silencing. UHRF1 overexpression, as well as PPARG-silencing, imparts higher growth rate and phenotypic features resembling those occurring in the epithelial-mesenchymal transition. In our series of 110 sporadic CRCs, high UHRF1-expressing tumors are characterized by an undifferentiated phenotype, higher proliferation rate and poor clinical outcome only in advanced stages III-IV. In addition, the inverse relationship with PPARG found in vitro is detected in vivo and UHRF1 prognostic significance appears closely related to PPARG low expression, as remarkably validated in an independent dataset. The results demonstrate that UHRF1 regulates PPARG silencing and both genes appear to be part of a complex regulatory network. These findings suggest that the relationship between UHRF1 and PPARG may have a relevant role in CRC progressio